Anti-TSLP antibody compositions and uses thereof

Abstract

The present application relates generally to compositions comprising the anti-TSLP antibody tezepelumab and its derivatives, which have quality attributes of the antibody.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 178,938, filed April 23, 2021, the entirety of which is incorporated herein by reference.

[0002] The present application relates generally to compositions comprising the anti-TSLP antibody, tezepelumab, and its derivatives, including quality attributes of the antibody. [Background technology]

[0003] Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine produced in response to environmental and proinflammatory stimuli, leads to activation of multiple inflammatory cells and downstream pathways (Soumelis et al. Nat Immunol 2002;3:673-80; Allakhverdi et al. J Exp Med 2007;204:253-8). TSLP is increased in the airways of asthmatics and correlates with expression of Th2 cytokines and chemokines (Shikotra et al. J Allergy Clin Immunol 2012;129:104-11 e1-9) and disease severity (Ying et al. J Immunol 2005;174:8183-90; Ying et al. J Immunol 2008;181:2790-8). While TSLP is central to regulating Th2 immunity, it may also play an important role in other pathways of inflammation and thus may be associated with multiple asthma phenotypes.

[0004] Tezepelumab is a human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to TSLP and prevents its interaction with the TSLP receptor complex. Tezepelumab is a heterotetramer composed of two heavy chains and two light chains, and contains two binding sites for TSLP. In a proof-of-concept study in patients with mild atopic asthma, tezepelumab was shown to inhibit early and late asthmatic responses and suppress biomarkers of Th2 inflammation after inhaled allergen exposure (Gauvreau,et al.N Engl J Med 2014;370:2102-10). Summary of the Invention [Means for solving the problem]

[0005] Monitoring antibody therapeutics in formulations over time is important to identify storage conditions that reduce any degradation of the therapeutic and maintain product integrity. The present disclosure provides testing of anti-TSLP antibody attributes that may change over time during manufacture and storage, including attributes that may be beneficial or detrimental to the tolerability and / or efficacy of the antibody.

[0006] In one aspect, the disclosure provides a composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise an isomerized derivative, and the amount of the isomerized derivative in the composition is less than about 30%, and wherein the tezepelumab comprises: (A) a light chain variable domain comprising: (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:8.

[0007] In various embodiments, the amount of isomerized derivative in the composition is less than about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In various embodiments, the amount of isomerized derivative in the composition is about 0.5% to about 13%. In various embodiments, the amount of isomerized derivative in the composition is about 1% to 12%, 2% to 10%, or about 4% to 7%. In various embodiments, the isomerized derivative comprises a modification in the heavy or light chain complementarity determining region (CDR). In various embodiments, the isomerized derivative comprises a mutation in one or both of the variable region chains at heavy chain CDR residue D54 of SEQ ID NO: 7, and / or light chain CDR residues D49, D50, or D52 of SEQ ID NO: 4. In various embodiments, the isomerized derivative comprises an isomerization at D54 in an amount less than about 5%. In various embodiments, the isomerized derivative comprises isomerization at D54 in an amount of less than about 4%, 3%, 2%, or 1%. In various embodiments, the isomerized derivative comprises isomerization at one or more of residues D49, D50, or D52 of SEQ ID NO:4 in an amount of less than about 26%. In various embodiments, the isomerized derivative comprises isomerization at one or more of residues D49, D50, or D52 of SEQ ID NO:4 in an amount of less than about 25%, 20%, 18%, 15%, 13%, 10%, 7%, 5%, 3%, or 2%. In various embodiments, the isomerized derivative is isoaspartic acid (isoAsp), cyclic aspartic acid (cAsp), succinimide, or an isomerized intermediate. In various embodiments, the isomerized derivative is isoaspartic acid (isoAsp) or cyclic aspartic acid (cAsp). In various embodiments, the amount of isomerized derivative in the composition is measured by reduced peptide mapping. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions containing greater than 30% isomerized derivatives, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads.In various embodiments, tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions comprising greater than 30% isomerized derivatives, said efficacy including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0008] A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a deamidated derivative, and the amount of the deamidated derivative in the composition is less than about 15%, wherein the tezepelumab comprises: (A) a light chain variable domain comprising (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO: 3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO: 4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO: 5; and (B) a heavy chain variable domain comprising (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO: 6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO: 8. In various embodiments, the amount of the deamidated derivative in the composition is less than about 15%, about 10%, about 7%, about 5%, about 4%, about 3%, about 2%, or about 1%. In various embodiments, the amount of the deamidated derivative in the composition is about 0.5% to about 13%. In various embodiments, the amount of deamidated derivative in the composition is about 0.5%-10%, about 1%-8%, about 2%-7%, or about 3%-6%. In various embodiments, the deamidated derivative comprises a deamidated asparagine residue at N25 / N26 of LCDR1 set forth in SEQ ID NO:3, N316 of the heavy chain set forth in SEQ ID NO:13, and / or N385 / 390 of the heavy chain set forth in SEQ ID NO:13. In various embodiments, the deamidated derivative comprises deamidation at N25 / N26 in an amount of less than about 3%. In various embodiments, the deamidated derivative comprises deamidation at one or more of N316 and / or N385 / 390 in an amount of less than about 13%. In various embodiments, the deamidated derivative is a deamidated asparagine or a deamidated intermediate. In various embodiments, the amount of deamidated derivative in the composition is measured by reduced peptide mapping. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 15% deamidated derivatives, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads.In various embodiments, tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions comprising greater than 15% deamidated derivatives, said efficacy including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to the TSLPR.

[0009] The disclosure also provides compositions comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise an oxidized derivative, and the amount of oxidized derivatives in the composition is less than about 7%, and wherein the tezepelumab comprises: (A) a light chain variable domain comprising: (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:8.

[0010] In various embodiments, the amount of oxidized derivative is less than about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%. In various embodiments, the amount of oxidized derivative in the composition is about 0.4% to about 7%, about 1% to about 6%, about 2% to about 5%, or about 0.4% to about 4%. In various embodiments, the oxidized derivative comprises an oxidation of one or more of heavy chain methionine residue M34 of HCDR1 set forth in SEQ ID NO:6, or heavy chain methionine residues M253 or M359 of the heavy chain constant region set forth in SEQ ID NO:13, or heavy chain tryptophan residue W52 of HCDR2 set forth in SEQ ID NO:7, W90 of LCDR3 set forth in SEQ ID NO:5, or W102 of HCDR3 set forth in SEQ ID NO:8, in one or both of the heavy chains. In various embodiments, the oxidized derivative comprises oxidation of one or more of heavy chain methionine residues M34, M253, M359 in one or both of the heavy chains, optionally in an amount of less than about 7%. In various embodiments, the oxidized derivative comprises oxidation of one or more of tryptophan residues W52, W90, or W102 in one or both of the heavy chains, optionally in an amount of less than about 3%. In various embodiments, the amount of oxidized derivative in the composition is measured by reduction peptide mapping. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 7% oxidized derivative, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 7% oxidized derivatives, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, expression of which indicates that TSLP binds to TSLPR.

[0011] In another embodiment, the disclosure provides a composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a glycosylated derivative, and the amount of glycosylated derivatives in the composition is less than about 40%, and wherein the tezepelumab comprises: (A) a light chain variable domain comprising: (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:8.

[0012] In various embodiments, the amount of glycosylated derivatives in the composition is less than about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, or about 5%. In various embodiments, the amount of glycosylated derivatives in the composition is about 1% to about 35%, about 3% to about 30%, about 5% to about 25%, about 10% to about 20%. In various embodiments, the glycosylated derivative comprises a glycosylation mutation of tezepelumab at residue N298 of SEQ ID NO: 13 in one or both heavy chains. In various embodiments, the glycosylated derivative comprises afucosylation of tezepelumab or a mutation of glycosylation to a high mannose moiety or a galactosyl moiety. In various embodiments, the glycosylated derivative comprises an amount of afucosylated derivatives less than about 5%. In various embodiments, the glycosylated derivatives contain less than about 30% galactosyl moieties. In various embodiments, the glycosylated derivatives contain less than about 5% high mannose moieties. In various embodiments, the amount of glycosylated derivatives in the composition is measured by glycan mapping. In various embodiments, the tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions containing more than 40% glycosylated derivatives, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, the tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions containing more than 40% glycosylated derivatives, said potency including the ability to inhibit the binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0013] Glycosylation derivatives can also be associated with effector function and antibody clearance (it will be understood that lower antibody clearance can indicate longer half-life. Thus, it will be understood that an antibody or antibody composition that has "lower clearance" than a reference antibody or antibody composition means that it has a numerically longer half-life than the reference antibody or antibody composition). In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or are better tolerated than compositions comprising more than about 15%, about 13%, about 11%, about 8% or about 6% high mannose glycosylation derivatives. In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or are better tolerated than compositions comprising more than about 25%, about 23% (e.g., about 23.1%), about 21%, about 18%, about 15%, about 13%, about 11%, about 8%, about 6%, or about 5% high mannose glycosylated derivatives. In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or are better tolerated than compositions comprising more than about 23.1% high mannose glycosylated derivatives. In various embodiments, increasing the high mannose species from 5% to 23.1% results in an increase in clearance of tezepelumab and tezepelumab derivatives of 1.7% or less than 10%.

[0014] Also provided are compositions comprising tezepelumab and one or more disulfide isoform derivatives thereof, wherein the one or more disulfide isoform derivatives comprise an IgG2-B isoform and / or an IgG2-A / B isoform, and the amount of disulfide isoforms in the composition is less than about 75%. In various embodiments, the amount of disulfide isoforms in the composition is less than about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0015] In various embodiments, the one or more disulfide isoform derivatives comprise an IgG2-B isoform. In various embodiments, the amount of IgG2-B isoform is less than about 5%. In various embodiments, the one or more disulfide isoform derivatives comprise an IgG2-A / B isoform. In various embodiments, the amount of IgG2-A / B isoform in the composition is less than about 75%. In various embodiments, the amount of IgG2-A / B isoform in the composition is about 38% to about 43%. In various embodiments, the amount of disulfide isoform derivatives in the composition is measured by non-reducing reversed-phase high performance liquid chromatography (RP-HPLC).

[0016] Also contemplated are compositions comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives are high molecular weight (HMW) species, and the amount of HMW species in the composition is less than about 20%, and wherein the tezepelumab comprises: (A) a light chain variable domain comprising: (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:8.

[0017] In various embodiments, the amount of HMW species in the composition is less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In various embodiments, the amount of HMW species in the composition is about 0.5% to about 13%, about 1% to about 11%, about 2% to about 10%, or about 3% to about 8%, or about 4% to about 7%. In various embodiments, the amount of HMW species in the composition is about 1.7% or less. In various embodiments, the amount of HMW species in the composition is about 1.4% or less. In various embodiments, the HMW species comprises a dimer of tezepelumab.

[0018] In various embodiments, the amount of HMW species in the composition is measured by size-exclusion high performance liquid chromatography (SE-HPLC), sedimentation velocity ultracentrifugation (SV-AUC), or reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS). In various embodiments, the SE-HPLC is SE-Ultra HPLC (SE-UHPLC) or SE-HPLC with static light scattering (SE-HPLC-SLS). In various embodiments, the SE-HPLC is SE-UHPLC. In various embodiments, when the SE-HPLC is SE-UHPLC, the proteins are separated isocratically using a mobile phase comprising 100 mM sodium phosphate, 250 mM sodium chloride at pH 6.8. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 20% HMW species, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions containing greater than 20% HMW species, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0019] In various embodiments of the compositions, (a) tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions comprising more than 40% glycosylated derivatives, said efficacy including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads, or the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR, or (b) tezepelumab and tezepelumab derivatives comprise 15% or less high mannose and have lower clearance than compositions having more than 15% high mannose.

[0020] In various embodiments of the compositions, (a) tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions comprising more than 40% glycosylated derivatives, said efficacy including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads, or the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR, or (b) tezepelumab and tezepelumab derivatives comprise 25% or less high mannose and have lower clearance than compositions having more than 25% high mannose.

[0021] Also provided are compositions comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a tezepelumab fragment, and the amount of the tezepelumab fragment in the composition is less than about 15%, and the tezepelumab comprises a light chain variable domain comprising: (i) a light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:8.

[0022] In various embodiments, the amount of fragments in the composition is less than about 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In various embodiments, the amount of fragments in the composition is about 0.5% to about 13%, about 1% to about 11%, about 2% to about 10%, or about 3% to about 8%, or about 4% to about 7%. In various embodiments, the tezepelumab fragments are low molecular weight (LMW) or medium molecular weight (MMW) species, or a combination thereof. In various embodiments, the fragments are low molecular weight species less than about 25 kD. In various embodiments, the fragments are medium molecular weight species having a molecular weight of about 25 to 50 kD. In various embodiments, the amount of tezepelumab fragments in the composition is measured by reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS). In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 20% tezepelumab fragments, said potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 15% tezepelumab fragments, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0023] Provided herein are compositions comprising tezepelumab and one or more tezepelumab derivatives, including isomerized derivatives, deamidated derivatives, oxidized derivatives, glycosylated derivatives, HMW species, fragments, disulfide isomers, or combinations thereof, wherein the compositions have one or more of the following characteristics: (a) the amount of isomerized derivatives in the composition is about 30% or less as measured by reduced peptide mapping; (b) the amount of deamidated derivatives in the composition is about 15% or less as measured by peptide mapping; (c) the amount of oxidatively derived derivatives in the composition is about 15% or less as measured by reduced peptide mapping; (d) the amount of glycosylated derivatives in the composition is about 75% or less as measured by glycan mapping; (e) the amount of disulfide isomers in the composition is about 40% or less as measured by non-reducing reverse phase high performance liquid chromatography (RP-HPLC); (f) the amount of HMW species in the composition is about 20% or less as measured by SE-HPLC; and / or (g) the amount of fragments in the composition is about 20% or less as measured by rCE-SDS.

[0024] In various embodiments, tezepelumab comprises a heavy chain amino acid sequence set forth in SEQ ID NO:10 and a light chain amino acid sequence set forth in SEQ ID NO:12.

[0025] In another aspect, the disclosure provides a method for assessing the quality of a tezepelumab composition, comprising obtaining a tezepelumab composition containing tezepelumab and one or more tezepelumab derivatives; measuring the amount of one or more tezepelumab derivatives in the composition, the tezepelumab derivatives including isomerized derivatives, deamidated derivatives, oxidized derivatives, glycosylated derivatives, disulfide isoform derivatives, HMW species, fragments, or combinations thereof; comparing the measured amount of one or more tezepelumab derivatives to a predefined reference standard; and preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition if the comparison indicates satisfaction of the predefined reference standard. In various embodiments, the amount of isomerized derivatives is measured, and the predefined reference standard is about 30% or less. In various embodiments, the amount of isomerization in the tezepelumab composition is measured by reduced peptide mapping. In various embodiments, the amount of deamidated derivatives is measured, and the predefined reference standard is about 15% or less. In various embodiments, the amount of deamidation in the tezepelumab composition is measured by reduced peptide mapping. In various embodiments, the amount of oxidized derivatives is measured, the predetermined reference standard being about 7% or less. In various embodiments, the amount of oxidation in the tezepelumab composition is measured by reduced peptide mapping. In various embodiments, the amount of glycosylated derivatives is measured, the predetermined reference standard being about 40% or less. In various embodiments, the amount of glycosylation in the tezepelumab composition is measured by glycan mapping. In various embodiments, the amount of disulfide isoform derivatives is measured, the predetermined reference standard being about 75% or less. In various embodiments, the amount of disulfide isoforms in the tezepelumab composition is measured by non-reducing reversed-phase high performance liquid chromatography (RP-HPLC). In various embodiments, the amount of HMW species is measured, the predetermined reference standard being about 20% or less. In various embodiments, the amount of HMW species is measured by SE-HPLC. In various embodiments, the amount of fragments is measured, the predetermined reference standard being about 15% or less. In various embodiments, the amount of fragments in the tezepelumab composition is measured by rCE-SDS.

[0026] In various embodiments, the tezepelumab composition is obtained from a Chinese Hamster Ovary (CHO) cell line expressing a nucleic acid encoding a heavy chain of SEQ ID NO:10 and a nucleic acid encoding a light chain of SEQ ID NO:12.

[0027] In various embodiments, the immunoglobulin, antigen binding protein, or antibody is a human antibody. In various embodiments, the antibody is an IgG2 antibody. In various embodiments, the tezepelumab or derivatives thereof specifically bind to a TSLP polypeptide as set forth in amino acids 29-159 of SEQ ID NO:2. In various embodiments, both binding sites of the tezepelumab or derivatives thereof have the same binding to TSLP.

[0028] In various embodiments, tezepelumab or a derivative thereof is a compound having a molecular weight of 10 -8 Binds to TSLP with an affinity below the M Kd.

[0029] Additionally, compositions comprising tezepelumab or a derivative thereof as described herein and a pharma- ceutically acceptable carrier, excipient, or diluent are contemplated.

[0030] The present disclosure also provides an isolated nucleic acid comprising a polynucleotide sequence encoding the light chain variable domain, the heavy chain variable domain, or both, of tezepelumab or a derivative thereof described herein.

[0031] The disclosure further contemplates a recombinant expression vector comprising a nucleic acid encoding tezepelumab as described herein. Host cells comprising the expression vector are also provided.

[0032] Further contemplated herein is a method of producing a composition comprising tezepelumab or a derivative thereof that specifically binds to a TSLP polypeptide comprising amino acids 29-159 of SEQ ID NO:2, comprising incubating a host cell under conditions permitting expression of an immunoglobulin, antigen binding protein, or antibody, wherein the host cell comprises (i) a recombinant expression vector encoding a light chain variable domain of an antigen binding protein as described herein, and a recombinant expression vector encoding a heavy chain variable domain of an antigen binding protein as described herein, or (ii) a recombinant expression vector encoding both the light chain variable domain and the heavy chain variable domain of tezepelumab.

[0033] Also provided herein is a method for treating an inflammatory disease in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of tezepelumab and its derivatives. In various embodiments, the inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic idiopathic urticaria, Ig-induced disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF). In various embodiments, the asthma is mild, moderate, or severe asthma. In various embodiments, the asthma is severe asthma. In various embodiments, the asthma is eosinophilic asthma or non-eosinophilic asthma.

[0034] In various embodiments, the method comprises administering the composition at intervals of every two weeks or every four weeks, hi various embodiments, the composition is administered for a period of at least four months, six months, nine months, one year or more.

[0035] In various embodiments, the antibody is an IgG2 antibody. In various embodiments, the tezepelumab or tezepelumab derivative comprises a heavy chain variable region set forth in SEQ ID NO: 10 and a light chain variable region set forth in SEQ ID NO: 12, and includes one or more of the attributes described herein.

[0036] The present disclosure also provides compositions comprising tezepelumab and derivatives thereof as described herein for use in treating an inflammatory disease. In certain embodiments, the present disclosure provides the use of a composition comprising tezepelumab and derivatives thereof as described herein in the preparation of a medicament for treating an inflammatory disease.

[0037] Also contemplated is a syringe, for example a disposable or pre-filled syringe, a sterile sealed container, for example a vial, a bottle, a container, and / or a kit or package, which comprises any of the above-mentioned antibodies or compositions, optionally with suitable instructions for use.In various embodiments, administration is via a pre-filled syringe or an automatic injection device.In various embodiments, the automatic injection device is the Ypsomed YpsoMate® device.

[0038] It is understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention and is therefore intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described using terms such as "one embodiment," "some embodiments," "particular embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without the need to recite every possible combination. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any numerical values ​​between such endpoints are contemplated, with any combination of upper and lower endpoints being envisioned.

[0039] Headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments and variations of the invention will become apparent from the detailed description and / or drawings and / or claims. [Brief description of the drawings]

[0040] [Fig. 1A-1F] FIG. 1 is a series of leverage plots showing the relationship between potency and total CDR IsoAsp (FIGS. 1A and 1D), HMW species (FIGS. 1B and 1E), and total CDR Trp oxidation (FIGS. 1C and 1F). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The structure of tezepelumab has been elucidated by a variety of biological, biochemical, and biophysical techniques to provide an understanding of its structural and functional properties, as well as an assessment of important quality attributes.

[0042] Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions set forth below.

[0043] As used in the specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly dictates otherwise.

[0044] Unless otherwise defined, 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 disclosure belongs.

[0045] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which error depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" is placed before the first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each of the numbers in the series.

[0046] The term "inflammatory disease" refers to a medical condition involving abnormal inflammation caused by the immune system attacking its own cells or tissues, which can result in chronic pain, redness, swelling, stiffness, and damage to normal tissue. Inflammatory diseases include, for example, asthma, chronic peptic ulcer, tuberculosis, periodontitis, sinusitis, active hepatitis, ankylosing spondylitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), Crohn's disease, ulcerative colitis, osteoarthritis, atherosclerosis, systemic lupus erythematosus, atopic dermatitis, eosinophilic esophagitis (EoE), nasal polyps, chronic idiopathic urticaria, Ig-induced diseases (such as IgA nephropathy and lupus nephritis), eosinophilic gastritis, chronic sinusitis without nasal polyps, idiopathic pulmonary fibrosis (IPF), and the like. In an exemplary embodiment, the inflammatory disease is asthma, atopic dermatitis, or COPD. In an exemplary aspect, the inflammation is asthma, and in some cases, the asthma is severe asthma, eosinophilic asthma, non-eosinophilic asthma, or hypoeosinophilic asthma.

[0047] As used herein, the term "asthma" refers to allergic asthma, non-allergic asthma, eosinophilic asthma, and non-eosinophilic asthma.

[0048] As used herein, the term "allergic asthma" refers to asthma induced by one or more inhaled allergens. Such patients have positive IgE fluorescent enzyme immunoassay (FEIA) levels against one or more allergens that induce asthmatic reactions. Most allergic asthma is usually associated with Th2-type inflammation.

[0049] The term "non-allergic asthma" refers to patients who have low eosinophil counts, low Th2, or low IgE at the time of diagnosis. Patients with "non-allergic asthma" usually have a negative IgE fluorescent enzyme immunoassay (FEIA), which responds to an allergen panel that includes area-specific allergens. In addition to low IgE, these patients often have low or no eosinophil counts and low Th2 counts at the time of diagnosis.

[0050] As used herein, the term "severe asthma" refers to asthma that requires intensive treatment (e.g., GINA Steps 4 and 5) to maintain good control or where good control is not achieved despite intensive treatment (GINA, Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA) December 2012).

[0051] As used herein, the term "eosinophilic asthma" refers to asthma patients with blood eosinophil counts at screening of 300 cells / μL or less, or 250 cells / μL or less, and "hypoeosinophilic" asthma refers to asthma patients with eosinophil counts in blood or serum less than 250 cells / μL. Alternatively, "hypoeosinophilic" asthma refers to asthma patients with eosinophil counts in blood or serum less than 300 cells / μL.

[0052] "T helper (Th)1 cytokines" or "Th1 specific cytokines" refer to cytokines expressed (intracellularly expressed and / or secreted) by Th1 T cells, including IFN-g, TNF-a, and IL-12. "Th2 cytokines" or "Th2 specific cytokines" refer to cytokines expressed (intracellularly expressed and / or secreted) by Th2 T cells, including IL-4, IL-5, IL-13, and IL-10. "Th17 cytokines" or "Th17 specific cytokines" refer to cytokines expressed (intracellularly expressed and / or secreted) by Th17 T cells, including IL-17A, IL-17F, IL-22, and IL-21. Certain populations of Th17 cells express IFN-g and / or IL-2 in addition to the Th17 cytokines listed herein. Polyfunctional CTL cytokines include IFN-g, TNF-a, IL-2, and IL-17.

[0053] The term "specifically binds" refers to an antibody or polypeptide that is "antigen-specific" and is "selective binding agent", "specific binding agent", "specific" for an "antigen target" or is "immunoreactive" to an antigen and binds to a target antigen with higher affinity than other antigens of similar sequence. The agent is contemplated herein to specifically bind a target protein that is useful for identifying immune cell types, e.g., surface antigens (e.g., T cell receptor, CD3), cytokines (e.g., TSLP, IL-4, IL-5, IL-13, IL-17, IFN-g, TNF-a), and the like. In various embodiments, an antibody specifically binds to a target antigen but can cross-react with orthologs in closely related species, e.g., an antibody can bind to a human protein and also to a closely related primate protein. In various embodiments, an immunoglobulin, antigen-binding protein or fragment thereof, or an antibody or fragment thereof specific for TLSP is numerically 10 -8 In various embodiments, the anti-TSLP antibodies described herein bind with a Kd of at least 10 -8 M, 10 -9 M, 10-10 M, 10 -11 M, 10 -12 M, 10 -13 It binds with an affinity (Kd) of less than or equal to M.

[0054] The term "antibody" refers to a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable region and a constant region. "Heavy chain" and "light chain" refer to substantially full-length canonical immunoglobulin light and heavy chains (see, e.g., Immunobiology, 5th Edition (Janeway and Travers et al., Eds., 2001)). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0055] Antigen-binding proteins include antibodies, antibody fragments, and antibody-like proteins, which may have structural variations relative to the canonical tetrameric antibody structure. Antibody "variants" refer to antigen-binding proteins or fragments thereof that may have structural variations in antibody sequence or function compared to a parent antibody with a known sequence. Antibody variants include V regions with variations in the constant region, or alternatively, optionally adding V regions to the constant region in a non-canonical manner. Examples include multispecific antibodies (e.g., bispecific antibodies with additional V regions), antibody fragments capable of binding to antigen (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, diabodies), biparatopic peptides and recombinant peptides including the above, so long as they exhibit the desired biological activity.

[0056] Antibody fragments include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), complementarity determining region (CDR) fragments, CDR-grafted antibody binding regions, single chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelating recombinant antibodies, tri- or bibodies, intrabodies, nanobodies, small modular immunopreparations (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single domain antibodies (including camelized antibodies), VHH-containing antibodies, or variants or derivatives thereof, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, e.g., the antigen-binding portion of an antibody comprising one, two, three, four, five or six CDR sequences, so long as the antibody retains the desired biological activity.

[0057] As used herein, "antibody derivative" refers to an antibody, antigen-binding protein, or fragment thereof that can be characterized in terms of its chemical identity, chemical modification, or structural type (e.g., HMW species, fragment, or isoform) and contains one or more attributes described herein, which exhibit a desired biological activity.

[0058] "Valency" refers to the number of antigen-binding sites on each antibody or antibody fragment that target an epitope. A typical full-length IgG molecule, or F(ab)2, is "bivalent" in that it has two identical target binding sites. A "monovalent" antibody fragment, such as F(ab)' or scFc, has a single antigen-binding site. Trivalent or tetravalent antigen-binding proteins can also be engineered to be multivalent.

[0059] A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0060] The term "inhibiting TSLP activity" includes inhibiting any one or more of the following: binding of TSLP to its receptor; proliferation, activation, or differentiation of TSLPR-expressing cells in the presence of TSLP; inhibition of Th2 cytokine production in a polarization assay in the presence of TSLP; activation or maturation of dendritic cells in the presence of TSLP; and cytokine release of mast cells in the presence of TSLP. See, e.g., U.S. Pat. No. 7982016B2, line 6 and Example 8, and U.S. Patent Application Publication No. 2012 / 0020988A1, Examples 7-10.

[0061] The term "sample" or "biological sample" refers to a specimen obtained from a subject for use in the present methods, and includes urine, whole blood, plasma, serum, saliva, sputum, tissue biopsy, cerebrospinal fluid, peripheral blood mononuclear cells with in vitro stimulation, peripheral blood mononuclear cells without in vitro stimulation, intestinal lymphoid tissue with in vitro stimulation, intestinal lymphoid tissue without in vitro stimulation, intestinal lavage, bronchoalveolar lavage, nasal wash, and induced sputum.

[0062] The terms "treat", "treating" and "treatment" refer to the partial or complete elimination, reduction, inhibition or amelioration, either temporarily or permanently, of the clinical symptoms, onset or progression of an event, disease or condition associated with an inflammatory disorder as described herein. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given pathology, but need not necessarily eliminate every onset of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically need not necessarily be completely effective in preventing the onset of a condition to constitute a viable prophylactic agent. It is sufficient to merely reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by improving the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood that the disease will develop or worsen in the subject. One embodiment of the present invention relates to a method for measuring the effectiveness of a treatment, comprising administering a therapeutic agent to a patient in an amount and for a time sufficient to produce a sustained improvement over baseline in an indicator reflecting the severity of a particular disorder.

[0063] The term "therapeutically effective amount" refers to an amount of a therapeutic agent effective to ameliorate or alleviate a symptom or sign of a disease associated with a disease or disorder.

[0064] TSLP Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that is produced in response to proinflammatory stimuli and induces allergic inflammatory responses primarily through its activity on dendritic cells (Gilliet, J Exp Med. 197:1059-1067, 2003; Soumelis, Nat Immunol. 3:673-680, 2002; Reche, J Immunol. 167:336-343, 2001), mast cells (Allakhverdi, J Exp Med. 204:253-258, 2007), and CD34+ progenitor cells (Swedin et al. Pharmacol Ther 2017;169:13-34). TSLP signals through a heterodimeric receptor composed of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain and the common gamma chain-like receptor (TSLPR) (Pandey, Nat Immunol. 1:59-64, 2000; Park, J Exp Med. 192:659-669 (2000)).

[0065] Human TSLP mRNA (Brightling et al., J Allergy Clin Immunol 2008;121:5-10;quiz 1-2; Ortega et al. N Engl J Med 2014;371:1198-207) and protein levels (Ortega et al., supra) are increased in the airways of asthmatics compared to controls, and the magnitude of expression correlates with disease severity (Brightling et al., supra). Recent studies have shown an association between single nucleotide polymorphisms in the human TSLP locus and protection from asthma, atopic asthma, and airway hyperresponsiveness, suggesting that differential regulation of TSLP gene expression may affect disease susceptibility (Ortega et al. N Engl J Med 2014;371:1198-207; To et al. BMC Public Health 2012;12:204). These data suggest that targeting TSLP can inhibit multiple biological pathways involved in asthma.

[0066] Early preclinical studies of TSLP suggested that after release from airway epithelial or stromal cells, TSLP activates mast cells, dendritic cells, and T cells to release Th2 cytokines (e.g., IL-4 / 13 / 5). Recently published human data showed good correlation between tissue TSLP gene and protein expression, Th2 gene signature score, and tissue eosinophils in severe asthma. Thus, anti-TSLP targeted therapy may be effective in asthma patients with Th2-type inflammation (Shikotra et al, J Allergy Clin Immunol. 129(1):104-11, 2012).

[0067] Data from other studies suggest that TSLP may promote airway inflammation through Th2-independent pathways, such as crosstalk between airway smooth muscle and mast cells (Allakhverdi et al, J Allergy Clin Immunol. 123(4):958-60, 2009; Shikotra et al, supra). TSLP may also promote induction of T cells to differentiate into Th-17-cytokine producing cells, resulting in increased neutrophilic inflammation commonly seen in more severe asthma (Tanaka et al, Clin Exp Allergy. 39(1):89-100, 2009). These data, and other emerging evidence, suggest that blocking TSLP may play a role in suppressing multiple biological pathways, including but not limited to those involving Th2 cytokines (IL-4 / 13 / 5).

[0068] antibody It is contemplated that antibodies or antibody derivatives or antigen binding proteins specific for TSLP will be useful in the treatment of inflammatory diseases including asthma, e.g., severe asthma, eosinophilic asthma, noneosinophilic / hypoeosinophilic asthma and other forms of asthma described herein, atopic dermatitis, EoE, and COPD.

[0069] Specific binding agents, such as antibodies and antibody derivatives or fragments, that bind to a target antigen, e.g., TSLP, are useful in the methods and compositions of the disclosure. In one embodiment, the specific binding agent is an antibody. The antibody can be monoclonal (MAb); recombinant; chimeric; humanized, such as complementarity determining region (CDR) grafted; human; antibody variants, including single chain; and / or bispecific; as well as fragments, variants, or derivatives thereof. Antibody fragments include those portions of an antibody that bind to an epitope on a polypeptide of interest. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of a full-length antibody. Other binding fragments include fragments generated by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.

[0070] Monoclonal antibodies may be modified for use as therapeutic or diagnostic agents. One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and / or light (L) chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining portions of the chains are identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies are also included, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81:6851-55.

[0071] In another embodiment, the monoclonal antibody is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced from a source that is non-human. Humanization can be performed using methods described in the art, for example, by replacing at least a portion of a rodent complementarity determining region with the corresponding region of a human antibody (Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36).

[0072] Human antibody variants and derivatives (including antibody fragments) that bind TSLP are also encompassed by the present invention. Such antibodies are produced by immunization with a polypeptide antigen (i.e., having at least six consecutive amino acids), optionally conjugated to a carrier, using transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies without producing endogenous immunoglobulins. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. See also PCT Application Nos. PCT / US96 / 05928 and PCT / US93 / 06926. Further methods are described in U.S. Patent No. 5,545,807, PCT Application Nos. PCT / US91 / 245 and PCT / GB89 / 01207, and in EP 546073B1 and EP 546073A1. Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

[0073] Chimeric, CDR-grafted, and humanized antibodies, antibody fragments, and / or antibody variants and derivatives are typically produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is produced in a mammalian host cell, such as a CHO cell. Monoclonal (e.g., human) antibodies can be produced by expressing recombinant DNA in a host cell or by expressing in a hybridoma cell as described herein. Further examples of mammalian cells include Chinese hamster ovary (CHO) cells as well as immortalized cell lines available from the American Type Culture Collection (Manassas, VA), including HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and human epithelial kidney 293 cells. Additionally, cell lines or host systems can be selected to ensure correct modification and processing of tezepelumab or tezepelumab derivatives. Eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. These include CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030 and HsS78Bst cells. Human cell lines developed by immortalizing human lymphocytes can also be used. The human cell line PER.C6® (Janssen; Titusville, NJ) can also be used to recombinantly produce monoclonal antibodies.

[0074] By way of example, tezepelumab and tezepelumab derivatives having molecular attributes as described herein can be obtained by selecting cell clones expressing tezepelumab or tezepelumab derivatives having the molecular attributes. To produce such tezepelumab or tezepelumab derivatives, recombinant DNA methods can be used. For example, DNA encoding the heavy and light chains of tezepelumab or tezepelumab derivatives can be inserted into a suitable expression vector (or vectors, e.g., one vector for the heavy chain and one vector for the light chain), which can be transfected into a suitable host cell, e.g., a cell of a mammalian cell line. For example, suitable expression vectors containing a polynucleotide encoding a tezepelumab polypeptide linked to a promoter are known in the art. The expression vector can be introduced into a host cell by conventional techniques, and the transfected cell can be used to produce the antibody. Optionally, the host cell can be engineered to modulate the molecular attributes. For example, to modulate fucosylation, glycosylation-competent cells may be genetically modified to alter the activity of fucosyltransferases or Golgi GDP-fucose transporters. By way of example, engineering of cell lines to modulate glycosylation is described in PCT Publication WO 2015 / 116315.

[0075] Clones producing tezepelumab or tezepelumab derivatives containing the relevant molecular attributes may be selected. By way of example, established microtiter plate-based clone generation and expansion methods may be implemented. Hundreds of pooled heterogeneous cells may be sorted into single cell cultures by processes such as fluorescence activated cell sorting (FACS) or limiting dilution. After allowing a recovery period for healthy and stable populations, these clone-derived cells may be analyzed and select populations are selected for further analysis. For further analysis, clonal cells are cultured in "small-scale cell cultures" (e.g., a 10-day fed-batch process) in small vessels such as spin tubes, 24-well plates, or 96-deep well plates. In this small-scale process, boluses of nutrients are added periodically to obtain different cell growth and viability measurements. Hundreds or thousands of these small-scale cultures may be performed in parallel. At the end of the culture (e.g., day 10), cells are harvested for assay and analysis. Optionally, microtiter plate-based methods of generating and expanding clones (e.g., subcloning) may be implemented using, for example, microtiter plate-based methods developed by Berkeley Lights Beacon. (商標) This may be substituted with the use of automated or partially automated high-throughput and high-content screening tools, such as photoelectron cell line generation and analysis systems. Optionally, high-throughput screening methods and machine learning tools may be used to facilitate the selection of clones that give rise to relevant molecular attributes (see, for example, PCT Publication WO 2020 / 223422).

[0076] The anti-TSLP antibody tezepelumab is described in U.S. Pat. No. 7,982,016 and U.S. patent application Ser. No. 15 / 951,602.

[0077] Anti-TSLP antigen-binding proteins (including fragments thereof) useful in the present methods include anti-TSLP antibodies composed of: a. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; iii. a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and bi. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and iii. a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the antibody or antibody derivative specifically binds to a TSLP polypeptide as set forth in amino acids 29 to 159 of SEQ ID NO:2.

[0078] Also contemplated is an antibody or antibody derivative comprising: ai an amino acid sequence that is at least 80% identical to SEQ ID NO:12; ii an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:11; iii. a light chain variable domain selected from the group consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:11; and bi an amino acid sequence that is at least 80% identical to SEQ ID NO:10; ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:9; iii. a heavy chain variable domain selected from the group consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:9; or c. a light chain variable domain of (a) and a heavy chain variable domain of (b), wherein the antibody or antibody derivative specifically binds to a TSLP polypeptide as set forth in amino acids 29 to 159 of SEQ ID NO:2.

[0079] Tezepelumab is an exemplary anti-TSLP antibody having ai. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; iii. a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and bi. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and iii. a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8.

[0080] Tezepelumab also comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:12, which is encoded by the polynucleotide sequence set forth in SEQ ID NO:11; and a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:10, which is encoded by the polynucleotide sequence set forth in SEQ ID NO:9.

[0081] In various embodiments, the anti-TSLP antibody or antibody derivative thereof is bivalent and is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0082] In various embodiments, the anti-TSLP antibody derivative is selected from the group consisting of a diabody, a triabody, a tetrabody, a Fab fragment, a single domain antibody, and an scFv, and the dosage is adjusted so that the binding sites are equimolar to that administered by a bivalent antibody.

[0083] The antibody or antibody derivative is intended to be an IgG2 antibody. An exemplary sequence of the constant region of human IgG2 is available from the Uniprot database under Uniprot number P01859, and is incorporated herein by reference. Information including sequence information on the heavy and light chain constant regions of other antibodies is also publicly available through the Uniprot database as well as other databases familiar to those skilled in the art of antibody engineering and antibody production. Tezepelumab is an IgG2 antibody. The full-length heavy and light chain sequences of Tezepelumab, including the IgG2 chain, are set forth in SEQ ID NOs: 13 and 14, respectively.

[0084] In certain embodiments, antibody derivatives include tetrameric glycosylated antibodies in which the number and / or type of glycosylation sites have been altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the variants contain more or fewer N-linked glycosylation sites than the native protein. Alternatively, substitutions that delete this sequence remove existing N-linked carbohydrate chains. They also result in rearrangement of N-linked carbohydrate chains, deleting one or more N-linked glycosylation sites (typically naturally occurring ones) and generating one or more new N-linked sites. Further antibody variants include cysteine ​​variants in which one or more cysteine ​​residues have been deleted or replaced with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be useful when the antibody must be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein, and usually have an even number of cysteine ​​residues to minimize interactions resulting from unpaired cysteines.

[0085] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify residues of antibodies that are important for human TSLP or to increase or decrease the affinity of the antibodies described herein for human TSLP.

[0086] According to certain embodiments, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity, (4) inhibit the formation of high molecular weight (HMW) species, and / or (5) confer or modify other physiochemical or functional properties in such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made within the native sequence (in certain embodiments, portions of the polypeptide outside of the domains that establish intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically cannot substantially alter the structural features of the parent sequence (e.g., the substituted amino acid should not tend to disrupt helices present in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized by those of skill in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0087] Method of preparation The tezepelumab compositions of the disclosure can be prepared by recombinantly expressing nucleic acids encoding the heavy and light chains in a host cell, partially purifying or purifying tezepelumab from the host cell culture or a host cell lysate, and assaying the resulting composition for one or more of the tezepelumab derivatives detailed herein according to the methods described in more detail below.

[0088] To recombinantly produce tezepelumab or a tezepelumab derivative, one or more nucleic acids encoding a heavy chain (e.g., a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 10) and a light chain (e.g., a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12) are inserted into one or more expression vectors. The nucleic acids encoding the heavy chain and the light chain can be inserted into a single expression vector, or they can be inserted into separate expression vectors. As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences required for the expression of an operably linked coding sequence in a particular host cell. Expression vectors may include sequences that affect or control transcription, translation, and, if present, introns, RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence may also be optionally encoded by the expression vector and, if desired, operably linked to the coding sequence of interest to allow the expressed polypeptide to be secreted by the recombinant host cell for easier isolation of the polypeptide of interest from the cell. The vector may also include one or more selectable marker genes to facilitate selection of host cells into which the vector has been introduced. Exemplary nucleic acids encoding the heavy and light chains of tezepelumab as well as suitable signal peptide sequences and other components of expression vectors for recombinantly expressing tezepelumab are described in U.S. Patent No. 7,982,016, which is incorporated herein by reference in its entirety, and are set forth herein in SEQ ID NO:9 and SEQ ID NO:11.

[0089] After an expression vector has been constructed and one or more nucleic acid molecules encoding the heavy and light chain components of tezepelumab or a derivative thereof have been inserted into the appropriate sites of the vector, the completed vector may be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the expression vector of tezepelumab or a derivative thereof into a selected host cell may be performed by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will depend, in part, on the host cell type used. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989).

[0090] When cultured under appropriate conditions, the host cells will synthesize tezepelumab or a derivative thereof, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the producing host cells (if not secreted). Selection of an appropriate host cell will depend on a variety of factors, including the desired expression level, polypeptide modifications desired or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0091] Exemplary host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and the like. Examples of suitable host organisms include the genera Bacillus, such as B. marcescans, and Shigella, and Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of lower eukaryotic host microorganisms.However, Pichia, e.g. P. pastoris, Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, Schwanniomyces, e.g. Schwanniomyces occidentalis, Several other genera, species, and strains are generally available and useful herein, such as A. occidentalis, as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0092] Host cells for expressing glycosylated antibodies can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and mutants have been identified, as well as corresponding insect permissive host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfecting such cells are publicly available, for example, the L-1 mutant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0093] Vertebrate host cells are also suitable hosts, and the recombinant production of antibodies from such cells is routine. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (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); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC mammalian myeloma cells, and several other cell lines. CHO cells are preferred host cells in some embodiments for expressing tezepelumab or a derivative thereof.

[0094] To produce tezepelumab or its derivatives, host cells are transformed or transfected with the expression vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. The host cells used to produce tezepelumab or its derivatives can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing 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. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; or WO 87 / 00195 may be used as a culture medium for the host cells. Any of these media may contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin, arginine ... (商標) Nutrients such as nutrient solutions containing 0.1% to 10% ethanol, 0.1% to 10% ethanol, 0.2% to 10% ethanol, 0.3% to 10% ethanol, 0.4% to 10% ethanol, 0.5% to 10% ethanol, 0.6% to 10% ethanol, 0.7% to 10% ethanol, 0.8% to 10% ethanol, 0.9 ...

[0095] When the host cells are cultured, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the host cells are lysed (e.g., by mechanical shearing, osmotic shock, or enzymatic methods) and particulate debris (e.g., host cells and lysed fragments) are removed, for example, by centrifugation, microfiltration, or ultrafiltration. If the antibody is secreted into the culture medium, it can be separated from the host cells by centrifugation or microfiltration, and optionally subsequently concentrated by ultrafiltration. Tezepelumab or a derivative thereof can be further purified or partially purified using one or more chromatographic steps, such as, for example, affinity chromatography (e.g., protein A or protein G affinity chromatography), cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography.

[0096] Upon production or acquisition of a tezepelumab composition, the composition can be evaluated for the presence and amount of one or more tezepelumab derivatives described herein, including isomerized derivatives (including isomerized intermediates thereof), deamidated derivatives (including deamidated intermediates thereof), oxidized derivatives, glycosylated derivatives, disulfide isoform derivatives, and size derivatives (e.g., HMW species or fragments). Thus, the present disclosure includes a method for evaluating the quality of a tezepelumab composition, comprising obtaining a tezepelumab composition containing tezepelumab and one or more tezepelumab derivatives; measuring the amount of one or more tezepelumab derivatives in the composition; comparing the measured amount of one or more tezepelumab derivatives to a predetermined reference standard; and, if the comparison indicates satisfaction of the predetermined reference standard, preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition. In some embodiments, the method comprises one, two, three, four, five, six, or seven of: (1) measuring the amount of an isomerized derivative (including an isomerized intermediate thereof) in the composition, (2) measuring the amount of a deamidated derivative (including a deamidated intermediate thereof) in the composition, (3) measuring the amount of an oxidized derivative in the composition, (4) measuring the amount of a glycosylated derivative in the composition, (5) measuring the amount of a disulfide isoform derivative in the composition, (6) measuring the amount of a HMW species in the composition, and / or (7) measuring the amount of a fragment in the composition. In certain embodiments, all seven measurements are performed on a tezepelumab composition.

[0097] The predetermined reference standard for each tezepelumab derivative may be a threshold amount or range of amounts of the derivative that does not significantly affect the efficacy and / or tolerability of the tezepelumab composition, for example, for safety purposes during administration or to inhibit ligand-induced activation of the TSLP receptor. For example, the predetermined reference standard for each tezepelumab derivative may be any of the limits or ranges disclosed herein for each of the derivatives, where tezepelumab compositions having these limits / ranges of the derivative have comparable efficacy and / or tolerability to tezepelumab compositions evaluated in clinical trials and shown to have clinical efficacy. It is understood that the predetermined reference standard described herein may be established prior to initiating the method as described herein.

[0098] In certain embodiments of the method, if the amount of the tezepelumab derivative measured in the composition meets a predetermined reference standard, the tezepelumab composition is classified as acceptable and can proceed to the next step in the manufacturing or distribution process, such as by preparing a pharmaceutical formulation of the composition (e.g., by combining with one or more excipients or diluents); preparing a pharmaceutical product of the composition (e.g., by filling into a vial, syringe, autoinjector, or other container or delivery device); packaging the composition with instructions for use, diluents, and / or a delivery device; or releasing the composition for marketing or distribution to a distributor. In some embodiments of the method, a pharmaceutical formulation of the tezepelumab composition is prepared if the amount of the tezepelumab composition measured in the composition meets a predetermined reference standard. In other embodiments of the method, a pharmaceutical product of the tezepelumab composition is prepared if the amount of the tezepelumab composition measured in the composition meets a predetermined reference standard. Methods for preparing pharmaceutical formulations and pharmaceutical products of the tezepelumab composition are described in more detail below. If the measured amount of tezepelumab derivative in the composition does not meet the predefined reference standard, in some embodiments of the method, the tezepelumab composition is classified as unacceptable and can be discarded or disposed of, or can be subjected to additional manufacturing steps, e.g., additional purification, to remove or reduce the amount of tezepelumab derivative in the composition such that the predefined reference standard is met.

[0099] In one embodiment, the method for evaluating the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and tezepelumab isomerized derivatives (including isomerized intermediates thereof); measuring the amount of isomerized derivatives in the composition; comparing the measured amount of isomerized derivatives with a predetermined reference standard; and, if the comparison indicates that the predetermined reference standard is met, preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition. The predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition can be less than about 30%, for example, less than about 25%, about 20% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, or about 4% or less. In one embodiment, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition is about 15% or less. In another embodiment, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition is about 13% or less. In another embodiment, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition is less than about 10%, about 8%, about 5%, about 3%, or about 2%. In some embodiments, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition can be a range of amounts, for example, about 0.5% to about 13% of the tezepelumab composition, about 1% to about 10% of the tezepelumab composition, or about 0.5% to about 5% of the tezepelumab composition. In another embodiment, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition is less than about 5%, 4%, 3%, 2%, or 1% isomerization at D54 of SEQ ID NO: 7 in one or both of the variable region chains. In various embodiments, the predetermined reference standard for the amount of isomerized derivatives in the tezepelumab composition is less than about 13%, about 10%, about 8%, about 5%, about 3%, or about 2% isomerization at one or more of D49, D50, or D52 of SEQ ID NO: 4. In certain embodiments, the amount of isomerized derivatives in the tezepelumab composition is measured by reduced peptide mapping.

[0100] In one embodiment, a method for evaluating the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and a tezepelumab deamidated derivative (including its deamidated intermediate); measuring the amount of the deamidated derivative in the composition; comparing the measured amount of the deamidated derivative with a predetermined reference standard; and, if the comparison indicates that the predetermined reference standard is met, preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition. The predetermined reference standard for the amount of the deamidated derivative in the tezepelumab composition can be less than about 15%, for example, about 13% or less, about 10% or less, about 8% or less, about 6% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. In one embodiment, the predetermined reference standard for the amount of the deamidated derivative in the tezepelumab composition is about 7% or less. In another embodiment, the predetermined reference standard for the amount of the deamidated derivative in the tezepelumab composition is about 5% or less. In another embodiment, the predetermined reference standard for the amount of deamidated derivatives in the tezepelumab composition is about 2% or less. In some embodiments, the predetermined reference standard for the amount of deamidated derivatives in the tezepelumab composition can be a range of amounts, for example, about 0.4% to about 10% of the tezepelumab composition, about 1% to about 7% of the tezepelumab composition, or about 0.1% to about 4% of the tezepelumab composition. In various embodiments, the predetermined reference standard for the amount of deamidated derivatives in the tezepelumab composition is less than about 3% deamidation at N25 / N26 of LCDR1 set forth in SEQ ID NO:3. In various embodiments, the predetermined reference standard for the amount of deamidated derivatives in the tezepelumab composition is less than about 13% deamidation at N316 of the heavy chain set forth in SEQ ID NO:13 and / or N385 / 390 of the heavy chain set forth in SEQ ID NO:13. In certain embodiments, the amount of deamidated derivatives in the tezepelumab composition is measured by reduced peptide mapping.

[0101] In one embodiment, the method for evaluating the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and tezepelumab oxidized derivatives; measuring the amount of oxidized derivatives in the composition; comparing the measured amount of oxidized derivatives with a predetermined reference standard; and, if the comparison indicates that the predetermined reference standard is met, preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition. The predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition can be less than about 7% or less, for example, about 6% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. In one embodiment, the predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition is about 7% or less. In another embodiment, the predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition is about 5% or less. In another embodiment, the predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition is about 3% or less. In some embodiments, the predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition can be a range of amounts, such as about 0.1% to about 7% of the tezepelumab composition, about 0.4% to about 5% of the tezepelumab composition, or about 0.8% to about 3% of the tezepelumab composition. In another embodiment, the predetermined reference standard for the amount of oxidized derivatives in the tezepelumab composition is about 7% or less, or about 6% or less, or about 5% or less, or about 3% or less oxidation at W102 of HCDR3 set forth in SEQ ID NO: 8. In certain embodiments, the amount of oxidized derivatives in the tezepelumab composition is measured by reduced peptide mapping.

[0102] In one embodiment, a method for evaluating the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and tezepelumab glycosylated derivatives; measuring the amount of glycosylated derivatives in the composition; comparing the measured amount of glycosylated derivatives to a predetermined reference standard; and, if the comparison indicates that the predetermined reference standard is met, preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition. The predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition can be less than about 40%, for example, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, or about 4% or less. In one embodiment, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 30% or less. In another embodiment, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 20% or less. In another embodiment, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 15% or less. In another embodiment, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 10% or less. In some embodiments, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition can be a range of amounts, such as about 1% to about 40% of the tezepelumab composition, about 4% to about 30% of the tezepelumab composition, about 2% to about 20% of the tezepelumab composition, or about 5% to about 15% of the tezepelumab composition. In various embodiments, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 5% or less, or about 4% or less of high mannose glycosylation in the composition. In various embodiments, the predetermined reference standard for the amount of glycosylated derivatives in a tezepelumab composition is about 25% or less, about 23% or less (e.g., about 23.1% or less), about 21% or less, about 19% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, about 5% or less, or about 4% or less of high mannose glycosylation in the composition.In various embodiments, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 23.1% or less of high mannose glycosylation in the composition. In various embodiments, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 30% or less, about 25% or less, about 20% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 5% or less, or about 4% or less of galactosylation in the composition. In various embodiments, the predetermined reference standard for the amount of glycosylated derivatives in the tezepelumab composition is about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less of afucosylated glycosylation. In certain embodiments, the amount of glycosylated derivatives in the tezepelumab composition is measured by glycan mapping.

[0103] In one embodiment, a method for assessing the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and tezepelumab disulfide isoform derivatives; measuring the amount of disulfide isoform derivatives in the composition; comparing the measured amount of disulfide isoforms to a predetermined reference standard; and preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition if the comparison indicates that the predetermined reference standard is met. The predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition can be less than about 75%, for example, about 70% or less, about 65% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, or about 4% or less. In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 50% or less. In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 35% or less. In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 25% or less. In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 15% or less. In another embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 10% or less. In another embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 8% or less. In some embodiments, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition can be a range of amounts, such as about 10% to about 70% of the tezepelumab composition, about 15% to about 50% of the tezepelumab composition, or about 20% to about 40% of the tezepelumab composition. In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 50% or less of the IgG2-A / B isoform.In one embodiment, the predetermined reference standard for the amount of disulfide isoform derivatives in the tezepelumab composition is about 5% or less of the IgG2-B isoform. In a particular embodiment, the amount of disulfide isoform derivatives in the tezepelumab composition is measured by reverse-phase HPLC.

[0104] In another embodiment, a method for assessing the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and HMW species of tezepelumab; measuring the amount of HMW species in the composition; comparing the measured amount of HMW species to a predefined reference standard; and preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition if the comparison indicates that the predefined reference standard is met. The predefined reference standard for the amount of HMW species in the tezepelumab composition can be less than about 20%, e.g., about 15% or less, about 12% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, or about 4% or less. The predetermined reference standard for the amount of HMW species in the tezepelumab composition can be less than about 3.0%, for example, about 2.5% or less, about 2.4% or less, about 2.3% or less, about 2.2% or less, about 2.1% or less, about 2.0% or less, about 1.8% or less, about 1.6% or less, about 1.4% or less, about 1.2% or less, about 1.0% or less, about 0.8% or less, about 0.6% or less, or about 0.4% or less. In one embodiment, the predetermined reference standard for the amount of HMW species in the tezepelumab composition is about 2.5% or less. In another embodiment, the predetermined reference standard for the amount of HMW species in the tezepelumab composition is about 1.7% or less. In another embodiment, the predetermined reference standard for the amount of HMW species in the tezepelumab composition is about 1.4% or less. In yet another embodiment, the predetermined reference standard for the amount of HMW species in the tezepelumab composition is about 1.2% or less. In yet another embodiment, the predetermined reference standard for the amount of HMW species in the tezepelumab composition is about 0.6% or less. The predetermined reference standard for the amount of HMW species in the tezepelumab composition can be a range of amounts in some embodiments, such as about 0.3% to about 2.4% of the tezepelumab composition, about 0.6% to about 2.1% of the tezepelumab composition, about 0.4% to about 1.2% of the tezepelumab composition, or about 0.6% to about 1.4% of the tezepelumab composition. In certain embodiments, the amount of HMW species in the tezepelumab composition is measured by SE-HPLC, e.g., SE-UHPLC, SE-HPLC-SLS, or sedimentation velocity ultracentrifugation (SV-AUC).

[0105] In another embodiment, a method for assessing the quality of a tezepelumab composition includes obtaining a tezepelumab composition containing tezepelumab and a fragment of tezepelumab (e.g., LMW or MMW); measuring the amount of the fragment in the composition; comparing the measured amount of the fragment to a predefined reference standard; and preparing a pharmaceutical formulation or pharmaceutical product of the tezepelumab composition if the comparison indicates that the predefined reference standard is met. The predefined reference standard for the amount of HMW species in the tezepelumab composition can be about 15% or less, about 12% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, or about 4% or less. The predetermined reference standard for the amount of fragments in the tezepelumab composition may be less than about 3.0%, for example, about 2.5% or less, about 2.4% or less, about 2.3% or less, about 2.2% or less, about 2.1% or less, about 2.0% or less, about 1.8% or less, about 1.6% or less, about 1.4% or less, about 1.2% or less, about 1.0% or less, about 0.8% or less, about 0.6% or less, or about 0.4% or less. In one embodiment, the predetermined reference standard for the amount of fragments in the tezepelumab composition is about 2.5% or less. In another embodiment, the predetermined reference standard for the amount of fragments in the tezepelumab composition is about 1.7% or less. In another embodiment, the predetermined reference standard for the amount of fragments in the tezepelumab composition is about 1.4% or less. In yet another embodiment, the predetermined reference standard for the amount of fragments in the tezepelumab composition is about 1.2% or less. In yet another embodiment, the predetermined reference standard for the amount of fragments in the tezepelumab composition is about 0.6% or less. The predetermined reference standard for the amount of fragments in the tezepelumab composition can be a range of amounts in some embodiments, such as about 0.3% to about 2.4% of the tezepelumab composition, about 0.6% to about 2.1% of the tezepelumab composition, about 0.4% to about 1.2% of the tezepelumab composition, or about 0.6% to about 1.4% of the tezepelumab composition. In certain embodiments, the amount of fragments in the tezepelumab composition is measured by rCE-SDS.

[0106] In certain embodiments of the methods of the present invention, the method comprises: (a) obtaining a tezepelumab composition containing tezepelumab and one or more tezepelumab derivatives, the tezepelumab derivatives including isomerized derivatives, deamidated derivatives, oxidized derivatives, glycosylated derivatives, disulfide isoform derivatives, HMW species, fragments, or combinations thereof; (b) evaluating the tezepelumab composition by performing one or more of the following: (i) determining the amount of isomerized derivatives in the composition by reduced peptide mapping and comparing the determined amount to a predetermined reference standard that is no greater than about 30%; (ii) determining the amount of the deamidated derivative in the composition by reduced peptide mapping and comparing the determined amount to a predetermined reference standard that is less than or equal to about 15%; (iii) determining the amount of oxidized derivatives in the composition by reduced peptide mapping and comparing the determined amount to a predetermined reference standard that is less than or equal to about 7%; (iv) determining the amount of glycosylated derivatives in the composition by glycan mapping and comparing the determined amount to a predetermined reference standard that is no greater than about 40%; (v) measuring the amount of disulfide isoform derivatives in the composition by non-reducing reversed-phase high performance liquid chromatography (RP-HPLC) and comparing the measured amount to a predetermined reference standard that is about 75% or less; (vi) determining the amount of HMW species in the composition by pre-peak in SE-HPLC and comparing the determined amount to a predetermined reference standard that is less than or equal to about 20%; and / or (vii) measuring the amount of the fragment in the composition by pre-peak in rCE-SDS and comparing the measured amount to a predetermined reference standard that is about 15% or less; and, (c) preparing a pharmaceutical formulation or product of the tezepelumab composition if the comparison in step (b) indicates satisfaction of the predetermined reference standard. In some embodiments, steps (b)(i), (b)(ii), (b)(iii), (b)(iv), (b)(v), (b)(vi), and (b)(vii) are all performed. In other embodiments, only steps (b)(vi) and (b)(vii) are performed. In certain embodiments, steps (b)(iv), (b)(vi), and (b)(vii) are performed.

[0107] Identifying attributes that contribute to protein binding To measure attributes that contribute to protein binding and activity, the anti-TSLP antibody tezepelumab as described herein is subjected to conditions that result in a change in its structure, e.g., the formation of a derivative of the therapeutic protein, resulting in a change in the amino acid structure of the therapeutic protein. In exemplary embodiments, the altered structure of an amino acid is referred to as an "attribute" and can be characterized in terms of its chemical identity or attribute type and its position in the amino acid sequence of the antigen binding protein (e.g., the position of the amino acid where the attribute is present). For example, asparagine and glutamine residues are susceptible to deamidation. A deamidated asparagine at position 10 of the amino acid sequence of a protein is an example of an attribute. A list of exemplary attribute types for certain amino acids is provided in Table A. Thus, as used herein, a "structure" can include, consist essentially of, or consist of the attribute types listed in Table A, or a combination of two or more attribute types listed in Table A. It will be understood that attributes are examples of structures, and that whenever "structure" is referred to herein, attributes are contemplated as examples of structures, unless otherwise indicated. For example, high molecular weight species (HMW) and fragments are also examples of attributes.

[0108] [Table A]

[0109] Since an immunoglobulin or fragment thereof, an antibody or antigen binding protein, such as tezepelumab, is composed of multiple amino acids, an antibody or antigen binding protein described herein may have more than one attribute (e.g., more than one amino acid with an altered structure) and may be described in terms of an attribute profile. As used herein, the term "attribute profile" refers to a list of attributes of an antigen binding protein. In various examples, the attribute profile optionally provides a chemical identity or attribute type, e.g., deamidation, relative to the native structure of the therapeutic protein. In various examples, the attribute profile provides the location of the attribute, e.g., the position of the amino acid at which the attribute is present. In some aspects, the attribute profile provides a description of all attributes present in the antigen binding protein. In other aspects, the attribute profile provides a description of some attributes present in the protein. For example, the attribute profile may provide only attributes present in a particular portion of the protein, e.g., the constant region, the variable region, the CDRs (such as the three light chain CDRs and the three heavy chain CDRs). A species of therapeutic protein, such as an antibody or antigen binding protein, is characterized by the attributes present in the protein. A species of antibody or antigen binding protein may differ from another species of the same protein by having a different attribute profile. If two therapeutic proteins have different attribute profiles, it indicates that the therapeutic proteins are two different species or derivatives of a therapeutic protein. If two therapeutic proteins have the same attribute profile, the therapeutic proteins are considered to be the same species or derivative of a therapeutic protein.

[0110] In various examples, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure (e.g., the formation of one or more attributes), which may alter the affinity of the therapeutic protein for the target. In various aspects, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure (e.g., the formation of one or more attributes), which may decrease the affinity of the antigen binding protein for the target. In some aspects, the decrease in affinity results in a partial or total loss of the ability of the immunoglobulin, antibody or antigen binding protein to interact (e.g., bind) with the target. In various examples, the partial or total loss of the ability of the immunoglobulin, antibody or antigen binding protein to interact (e.g., bind) with the target ultimately reduces the efficacy of the antigen binding protein. In an alternative example, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure (e.g., the formation of one or more attributes), which may not alter the affinity of the immunoglobulin, antibody or antigen binding protein for the target. In various aspects, the change in structure does not decrease the affinity of the protein for the target. Without being bound to any particular theory, the methods of the present disclosure advantageously accurately and precisely distinguish those attributes of an immunoglobulin, antibody or antigen binding protein that affect the interaction between an immunoglobulin, antibody or antigen binding protein and a target from those attributes that do not affect the interaction.

[0111] In various aspects, the compositions herein comprise a population of species or derivatives of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof. In various examples, the population is a homogenous population of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, and optionally, each of the proteins present in the composition sample is of the same species or derivative. In various examples, the population is a heterogeneous population composed of at least two different species or derivatives of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof having the attributes described herein. In various examples, the heterogeneous population is composed of at least two, at least three, at least four, at least five, at least six, at least seven or more different species or derivatives of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof. Optionally, the heterogeneous population is composed of more than seven, more than eight, more than nine, more than ten, more than twenty, more than thirty, more than forty, more than fifty different species or derivatives of proteins. In some aspects, each species or derivative of the population has a unique attribute profile. In representative examples, the species of immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof is the only protein present in the composition. In some aspects, the composition comprises (i) a population of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, and (ii) a pharma- ceutically acceptable carrier, diluent, excipient, or combination thereof. In some embodiments, at least 80%, 85%, 90%, 95%, or 99% of the immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof of the heterogeneous population comprise attributes as described herein. In some embodiments, no more than 20%, 15%, 10%, 5%, or 1% of the immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof of the heterogeneous population comprise attributes as described herein.

[0112] separation In exemplary embodiments, the disclosure includes methods for separating a mixture containing different species of antigens into at least two fractions. In some aspects, the mixture is separated into multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) fractions. In some aspects, the separation step of the methods disclosed herein preserves the native folding, conformation and binding ability of the antigen binding protein and its target. In various aspects, the mixture is separated into an unbound fraction, which contains unbound antibody or antigen binding protein or unbound target, and a bound fraction, which contains antibody / antigen binding protein-target complexes.

[0113] Suitable methods and techniques for separating mixtures into fractions are known in the art.See, for example, Coskun, North Clin Istanb 3(2);156-160(2016);Snyder et al., Practical HPLC Method Development, 2nd ed., John Wiley & Sons, Inc. 1997;Snyder et al., Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc., Hoboken, NJ, 2010;Heftmann, Chromatography: Fundamentals and applications of chromatography and related differential migration methods, 6th ed., Volume 69A, Elsevier, Amsterdam, Netherlands, 2004;Mori and Barth, Size Exclusion Chromatography, Springer-Verlag, Berlin, 1999. In some aspects, separation is based on charge, e.g., ion exchange chromatography, capillary isoelectric focusing (cIEF) and / or capillary zone electrophoresis (CZE), or based on hydrophobicity, e.g., separation in reversed phase (RP; e.g., RP-HPLC) and hydrophobic interaction chromatography (HIC-HPLC). In various aspects, separation is based on size, e.g., size exclusion chromatography (SEC; e.g., SE-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). The methods described herein are used to detect product oxidation of Met or Trp residues, fragmentation / clipping, isomerization of Asp, deamidation, formation of pyroglutamic acid at the N-terminus. In various embodiments, the mixture is separated into at least two fractions using a technique that separates components of the mixture based on size, charge, hydrophobicity, affinity for a capture molecule, or a combination thereof.In various examples, the technique is size exclusion chromatography (SEC), affinity chromatography, precipitation using beads or cells, free-flow fractionation (FFF), ion exchange chromatography (IEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), or ultracentrifugation (UC). Optionally, the mixture is separated into at least two fractions using a technique that separates components of the mixture based on size, optionally the technique is size exclusion chromatography (SEC).

[0114] In various embodiments, the mixture is separated into at least two fractions using a technique that separates the components of the mixture based on their affinity to a capture molecule bound to a solid support, optionally a bead or a cell. In various examples, the mixture is separated by (i) adding the mixture to a container, e.g., a tube, containing beads bound to the capture molecule or cells expressing the capture molecule on their surface, (ii) centrifuging the container (e.g., tube) to obtain a supernatant and a pellet, (iii) collecting the supernatant from the pellet to obtain an unbound fraction, (iv) releasing the bound fraction from the pellet with a solution, (v) centrifuging the container (e.g., tube) containing the pellet and the solution to obtain a second supernatant containing the bound fraction and a second pellet containing the beads or cells, and (vi) collecting the second supernatant to obtain a bound fraction. In some embodiments, the mixture is separated by (i) adding the mixture containing the beads bound to the capture molecule to a column to obtain a flow-through and a bound fraction, (ii) collecting the flow-through to obtain an unbound fraction, (iii) releasing the bound fraction from the beads with a solution and collecting a solution containing the bound fraction. Suitable solid supports include, for example, beads, resins, papers, optionally made of cellulose, silica, alumina, glass, plastic or combinations thereof. In an exemplary embodiment, the capture molecule bound to the solid support is a protein. The capture molecule may be the same as the target. Advantageously, the capture molecule is not limited to any particular molecule.

[0115] In various embodiments of the method of identifying attributes of an immunoglobulin, antigen binding protein (e.g., tezepelumab), or target that affect the interaction between an antigen binding protein and a target, the method comprises identifying and quantifying the abundance of each attribute present in the antigen binding protein species or derivative, or target, for each of the unbound and bound fractions, where if the abundance of the attribute in the unbound fraction is greater than the abundance of the attribute in the bound fraction, then the attribute adversely affects the interaction between the antigen binding protein and the target. In various aspects, the method comprises using a mass spectrometer to identify and quantify the abundance of each attribute of the antigen binding protein species or target in each of the unbound and bound fractions.

[0116] In various embodiments of the method of measuring the effect of a known attribute present in a species of antigen binding protein or target on the interaction between the antigen binding protein and the target, the method comprises quantifying the abundance of the known attribute for each of the unbound and bound fractions, where if the abundance of the known attribute in the unbound fraction is greater than the abundance of the known attribute in the bound fraction, the known attribute adversely affects the interaction between the antigen binding protein and the target. In various aspects, the method comprises quantifying the abundance of the known attribute in each of the unbound and bound fractions using a mass spectrometer.

[0117] Stability refers to resistance to chemical modifications of amino acid residues and biophysical protein modifications, such as the formation of HMW species during stress conditions that may occur during manufacturing, storage, and / or additional or alternative stress conditions. With respect to the methods of the embodiments described herein and immunoglobulins, antigen binding proteins, and fragments thereof, "stability" and / or "HMW" species may be measured using size exclusion chromatography (SEC). Compositions including immunoglobulins, antigen binding proteins, or fragments or derivatives may be separated by SEC, such as SEC-UV. SEC may use a mobile phase composed of 100 mM sodium phosphate and 250 mM NaCl (pH 6.8), a flow rate may be set at 0.5 ml / min, the column temperature may be set at 37° C., the run time may be 35 minutes, and the autosampler may be set at 4° C. An example of a column suitable for SEC includes a gel column (e.g., commercially available from TOSOH Bioscience as G3000SWxl column) containing silica particles containing diol functional groups and having an average diameter of 5 μm and an average pore size of about 25 nM. For SEC-UV, ultraviolet / visible spectroscopy (UV / VIS) detection can be performed at 214 nm and 280 nm. It will be appreciated that after separation, peaks representing monomeric and HMW species may elute at different times in the SEC elution profile.

[0118] After SEC analysis, peptide mapping may be optionally performed to identify peptide modifications associated with bound and unbound species, for example as described herein and / or in WO 2020 / 247790. For peptide mapping, a filter with a molecular weight cutoff (e.g., greater than 10 kDa) may be used to collect elution fractions and eluted with 7.5 M guanidine elution buffer. To identify chemical modifications that affect binding to antigen, stressed immunoglobulin (or antigen-binding protein or fragment thereof) and antigen may be mixed together and separated into antigen-binding complexes that are eluted first and unbound immunoglobulin (or antigen-binding protein or fragment thereof) that are eluted second. Monomeric and HMW species may be collected to identify chemical modifications that affect or correlate with HMW.

[0119] It will be understood that "affinity" or "binding" may also be measured by surface plasmon resonance (SPR), biolayer interferometry, or SEC binding affinity experiments as described herein. Unless otherwise specified herein or otherwise required by the scientific context, it will be understood that "affinity" refers to affinity as measured by SPR. Kd values ​​may be measured by SPR using a biosensor system such as the BIAcore® system. Analysis by the BIAcore® system may include analyzing the binding and dissociation of an antigen (e.g., TSLP) from a chip having immobilized molecules (e.g., anti-TSLP immunoglobulins, antigen binding proteins, or fragments thereof, as described herein) on its surface. Using SPR, a Kd<10 -6 Binding complexes of M can be detected. In various embodiments, SPR can be carried out at 20°C, 25°C, 30°C or 37°C.

[0120] composition It will be understood that the numbering of the residues in tezepelumab is based on the heavy and light chain variable sequences set forth in SEQ ID NOs: 10 and 12, respectively, and the full length antibody heavy and light chains set forth in SEQ ID NOs: 13 and 14, respectively.

[0121] In various embodiments, compositions are provided that include tezepelumab and one or more tezepelumab derivatives, each of which includes a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the derivatives include at least one of an isomerized derivative, a deamidated derivative, an oxidized derivative, a glycosylated derivative, a HMW species, a fragment, a disulfide isoform derivative, or a combination thereof. In various embodiments, compositions include tezepelumab and one or more tezepelumab derivatives, each of which includes a heavy chain amino acid sequence set forth in SEQ ID NO:10 and a light chain amino acid sequence set forth in SEQ ID NO:12.

[0122] The isomerized derivatives include mutations to aspartic acid residues. Exemplary isomerizations at aspartic acid include isoaspartic acid (isoAsp), cyclic aspartic acid (cAsp), succinimide, or other isomerization intermediates. The isomerized derivatives in the composition may include derivatives at the heavy or light chain complementarity determining regions (CDRs) or in other portions of the variable regions. In various embodiments, the isomerization is in the CDRs. The isomerized derivatives include mutations at D54 in the heavy chain CDR of SEQ ID NO: 7, and / or D49, D50, or D52 in the light chain CDR of SEQ ID NO: 4, in one or both of the variable region chains. In various embodiments, the amount of isomerized derivative in the composition is about 0.5% to about 30%, or about 0.5% to 13%. In some embodiments, the compositions include tezepelumab and derivatives thereof, and have an amount of isomerization at D54 of less than about 5% and / or an amount of isomerization at one or more of D49, D50, or D52 of less than about 13%. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions that include more than 30% isomerized derivatives, the potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions that include more than 30% isomerized derivatives, the potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0123] Deamidated derivatives include mutations to asparagine residues. Exemplary deamidated derivatives include complete deamidation and deamidated intermediates. The deamidated derivatives in the composition may include deamidated asparagine at N25 / N26 of LCDR1 set forth in SEQ ID NO:3, N316 of the heavy chain variable region set forth in SEQ ID NO:13, and / or N385 / 390 of the heavy chain variable region set forth in SEQ ID NO:13. In various embodiments, the composition includes a deamidated derivative that includes deamidation at N25 / N26 in an amount of less than about 3% and / or includes deamidation at one or more of N316 and / or N385 / 390 in an amount of less than about 13%. In some embodiments, the amount of deamidated derivatives in the composition is about 0.5% to 10%. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 15% deamidated derivatives, said potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 15% deamidated derivatives, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0124] Oxidized derivatives include mutations to one or more of the methionine or tryptophan residues in the protein. Exemplary oxidized derivatives include full oxidation or oxidation intermediates. The oxidized derivatives in the composition may include oxidation to one or more of the heavy chain methionine M34 of HCDR1 as set forth in SEQ ID NO:6, or M253 or M359 of the heavy chain constant region as set forth in SEQ ID NO:13, or heavy chain tryptophan W52 of HCDR2 as set forth in SEQ ID NO:7, W90 of LCDR3 as set forth in SEQ ID NO:5, or W102 of HCDR3 as set forth in SEQ ID NO:8, in one or both of the heavy chains (or light chains, if applicable). For example, the oxidized derivatives in the composition may include oxidation to one or more of the heavy chain methionine M34 of HCDR1 as set forth in SEQ ID NO:6, heavy chain tryptophan W52 of HCDR2 as set forth in SEQ ID NO:7, light chain W90 of LCDR3 as set forth in SEQ ID NO:5, or heavy chain W102 of HCDR3 as set forth in SEQ ID NO:8, in one or both of the heavy chains (or light chains, if applicable). In various embodiments, the oxidized derivative comprises oxidation of one or more of heavy chain methionines M34, M253, M359 in one or both of the heavy chains, optionally in an amount of less than about 7%. In various embodiments, the oxidized derivative comprises oxidation of one or more of tryptophans W52, W90, or W102 in one or both of the heavy chains, optionally in an amount of less than about 7%, optionally less than about 5%, or less than about 3%. In some embodiments, the amount of oxidized derivative in the composition is about 0.4% to about 7%. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 7% oxidized derivative, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 7% oxidized derivatives, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, expression of which indicates that TSLP binds to TSLPR.

[0125] High molecular weight derivatives include aggregation of antibodies into dimers or larger protein aggregates. HMW species contemplated herein include dimers and oligomers of tezepelumab. In various embodiments, the HMW species is a dimer. In various embodiments, the dimer is covalently or non-covalently linked. In various embodiments, the amount of HMW species in the composition is about 1.7% or less, about 1.6% or less, about 1.5% or less, about 1.4% or less, about 1.3% or less, about 1.2% or less, about 1.1% or less, about 1.0% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, or about 0.4% or less. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 20% HMW species, said potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 20% HMW species, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0126] Tezepelumab fragment derivatives include protein products that can be cleaved by internal peptidases produced during production or by other steps in the production process. Tezepelumab fragments include low molecular weight (LMW) species, e.g., less than about 25 kD, or medium molecular weight (MMW) species, e.g., 25-50 kD, or combinations thereof. In various embodiments, the amount of fragments in the composition is about 1.7% or less, about 1.6% or less, about 1.5% or less, about 1.4% or less, about 1.3% or less, about 1.2% or less, about 1.1% or less, about 1.0% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, or about 0.4% or less. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 15% fragment species, said potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 15% fragment species, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, expression of which indicates that TSLP binds to TSLPR.

[0127] Glycosylation derivatives of tezepelumab include alterations in the profile of sugar residues that may be post-translationally applied to asparagine residues in the Fc region of the antibody. Exemplary glycosylation derivatives include afucosylation, application of galactosyl moieties (galactosylation), and application of high mannose moieties to asparagine. Glycosylation derivatives contemplated herein are alterations to the sugar residue of asparagine N298 of the Fc region set forth in SEQ ID NO: 13 in one or both of the heavy chains. In various embodiments, the amount of glycosylation derivative in the composition is less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. In some embodiments, the glycosylation derivative comprises an afucosylation derivative in an amount of less than about 5%, about 4%, about 3%, or about 2%. In various embodiments, the glycosylated derivatives contain galactosyl moieties in an amount of less than about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. In various embodiments, the glycosylated derivatives contain high mannose moieties in an amount of about 25% or less, about 23% or less (e.g., about 23.1% or less), about 21% or less, about 19% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 5% or less, or about 4% or less. In various embodiments, the glycosylated derivatives contain high mannose moieties in an amount of about 23.1% or less. In various embodiments, the glycosylated derivatives contain high mannose moieties in an amount of about 5%, about 4%, about 3%, about 2%, or less than about 1%. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 40% glycosylated derivatives, said potency including the ability to inhibit binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 40% glycosylated derivatives, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.In various embodiments, tezepelumab and tezepelumab derivatives contain no more than 15%, 13%, 11%, 8%, or 5% high mannose and have a lower clearance (and / or longer half-life) than compositions with more than 15% high mannose. In various embodiments, tezepelumab and tezepelumab derivatives contain no more than about 25%, about 23%, about 21%, about 19%, about 17%, about 15%, about 13%, about 11%, about 8%, or about 5% high mannose and have a lower clearance (and / or longer half-life) than compositions with more than about 25% high mannose. In various embodiments, tezepelumab and tezepelumab derivatives contain no more than about 23.1% high mannose and have a lower clearance (and / or longer half-life) than compositions with more than about 23.1% high mannose. The percentage of high mannose can be measured by HILIC.

[0128] Tezepelumab or a tezepelumab derivative with "low clearance" refers to a low amount of clearance from the body (blood or serum) when compared to the clearance of a reference antibody, such as tezepelumab or other IgG2 antibodies. The clearance of tezepelumab or a tezepelumab derivative can be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or less clearance level compared to the reference antibody. The "long half-life" of tezepelumab or a tezepelumab derivative refers to the long time that the antibody is detectable in the body (blood or serum) when compared to the half-life of a reference antibody, such as tezepelumab or other IgG2 antibodies in the body. The half-life of tezepelumab or a tezepelumab derivative may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more longer than the half-life of the reference antibody.

[0129] In various embodiments, tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions containing more than about 40%, 35%, 30%, 25%, 23%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or 4% glycosylated derivatives.

[0130] The efficacy and / or tolerability of glycosylated derivatives may also be related to effector function and antibody clearance. In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or better tolerability than compositions comprising more than about 15%, about 13%, about 11%, about 8%, or about 6% high mannose glycosylated derivatives. In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or better tolerability than compositions comprising more than about 25%, about 23%, about 19%, about 17%, about 15%, about 13%, about 11%, about 8%, or about 6% high mannose glycosylated derivatives. In various embodiments, tezepelumab and tezepelumab derivatives have lower antibody clearance and / or better tolerability than compositions comprising more than about 23.1% high mannose glycosylated derivatives.

[0131] Disulfide structural heterogeneity is inherent in recombinant and naturally occurring IgG2 molecules, which contain 18 disulfide bonds (6 interchain and 12 intrachain disulfide bonds). In the classical IgG2-A structure, the hinge:hinge peptide contains four disulfide bonds. Unlike the classical IgG2-A structure, the isomeric IgG2-B contains two copies of the Fab peptide (C H 1-C L-hinge) with two copies of the hinge peptide. IgG2-A / B is an intermediate form that combines some features of both IgG2-A and IgG2-B and is defined by an asymmetric arrangement that includes one Fab arm covalently linked to two copies of the hinge peptide via a disulfide bond. Disulfide isoform derivatives are composed of IgG2-B isoforms and / or IgG2-A / B isoforms. In various embodiments, the amount of disulfide isoform derivatives in the composition is less than about 75%. In some embodiments, when the derivative is composed of IgG2-B isoforms, the amount of disulfide isoform derivatives in the composition is less than about 20%, about 15%, about 10%, or about 5%. In some embodiments, when the derivative is composed of the IgG2-A / B isoform, the amount of IgG2-A / B isoform in the composition is less than about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, or about 35%. In various embodiments, the amount of IgG2-A / B isoform in the composition is about 38% to about 43%. In various embodiments, the tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 75% disulfide isoform derivatives, said potency including the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads. In various embodiments, tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising greater than 75% disulfide isoform derivatives, said potency including the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0132] In various embodiments, the composition has one or more of the following characteristics: (a) the amount of isomerized derivative in the composition is about 30% or less as measured by reduced peptide mapping; (b) the amount of deamidated derivative in the composition is about 15% or less as measured by peptide mapping; (c) the amount of oxidized derivatives in the composition is about 7% or less as measured by reduced peptide mapping; (d) the amount of glycosylated derivatives in the composition is about 40% or less as determined by glycan mapping; (e) the amount of disulfide isoform derivatives in the composition is about 75% or less as measured by non-reducing reverse-phase high performance liquid chromatography (RP-HPLC); (f) the amount of HMW species in the composition is about 20% or less, as measured by SE-HPLC; and / or (g) the amount of fragments in the composition is about 15% or less as measured by rCE-SDS;

[0133] In some embodiments, the composition is part of a formulation described herein. In some embodiments, the composition is a drug substance used to produce a formulation as described herein.

[0134] Method of administration In one aspect, the methods of the disclosure comprise administering a therapeutic anti-TSLP antibody or antibody derivative described herein, optionally in a pharma- ceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is a sterile composition.

[0135] Contemplated herein are methods for treating inflammatory diseases, conditions, or disorders, such as asthma, chronic obstructive pulmonary disease (COPD), atopic dermatitis, eosinophilic esophagitis (EoE), nasal polyps, chronic idiopathic urticaria, Ig-induced disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF) with an anti-TSLP antibody or antigen binding protein or fragment thereof as described herein. In various embodiments, the disease, condition, or disorder is asthma, including severe asthma, eosinophilic asthma or non-eosinophilic asthma, and hypoeosinophilic asthma.

[0136] Asthma is a chronic inflammatory disorder of the airways. Each year, asthma causes an estimated 1.1 million outpatient visits, 1.6 million emergency department visits, 444,000 hospitalizations (Defrances et al, 2008, The Centers for Disease Control website, available at www.cdc.gov / nchs / data / nhsr / nhsr005.pdf), and 3,500 deaths in the United States. In susceptible individuals, asthmatic inflammation causes recurrent episodes of wheezing, shortness of breath, chest tightness, and coughing. The etiology of asthma is thought to be multifactorial, influenced by both genetic and environmental mechanisms (To et al., BMC Public Health 2012;12:204; Chung et al. Eur Respir J 2014;43:343-73), with environmental allergens being an important contributor (Chung et al., supra; Pavord ID, et al., NPJ Prim Care Respir Med 2017;27:17). The majority of cases arise when a person becomes hypersensitive to an allergen (atopy). Atopy is characterized by an increase in Th2 cells and expression of Th2 cytokines and production of IgE. In the United States, approximately 10 million patients are thought to suffer from allergy-induced asthma. Despite available treatment options, asthma remains a major health problem. Worldwide, approximately 300 million people currently suffer from asthma, and it is predicted that 400 million people will suffer from asthma by 2020 (Partridge, Eur Resp Rev. 16:67-72, 2007).

[0137] Allergen inhalation by atopic asthmatics induces some of the symptoms of asthma, including reversible airflow obstruction, airway hyperresponsiveness, and eosinophilic and basophilic airway inflammation. Allergen inhalation challenge has become the predominant model of asthma in many species (Bates et al., Am J Physiol Lung Cell Mol Physiol. 297(3):L401-10, 2009; Diamant et al., J Allergy Clin Immunol. 132(5):1045-1055, 2013).

[0138] Different asthma subtypes have been identified that are refractory to steroid treatment. Eosinophils are key inflammatory cells in allergic asthma, characteristically mediated by Th2-type CD4+ T cells. Neutrophilic airway inflammation is associated with corticosteroid treatment of severe asthma and can be mediated by Th1-type or Th17-type T cells (Mishra et al., Dis. Model. Mech. 6:877-888, 2013).

[0139] Diagnostic and assessment measures for asthma include: Airway inflammation assessed using a standardized single-breath exhaled nitric oxide (FeNO) (American Thoracic Society; ATS, Am J Respir Crit Care Med. 171(8):912-30, 2005) test. Spirometry is performed according to the ATS / European Respiratory Society (ERS) guidelines (Miller et al, Eur Respir J. 26(1):153-61, 2005). Post-bronchodilator (post-BD) spirometry test is assessed after subjects perform a pre-BD spirometry test. Maximal bronchodilation is induced using a SABA such as albuterol (90 μg metered dose) or salbutamol (100 μg metered dose) or equivalent spacer device for up to 8 total puffs (Sorkness et al, J Appl Physiol. 104(2):394-403, 2008). Maximal pre-BD and post-BD FEV1 obtained after 4, 6, or 8 puffs are used for determination and analysis of reversibility. The Asthma Control Questionnaire (ACQ) 6 is a patient-reported questionnaire that assesses asthma symptoms (i.e., night-time awakenings, symptoms on awakening, activity limitations, shortness of breath, wheezing) and daily rescue bronchodilator use and FEV1 (Juniper et al, Oct 1999). The ACQ-6 is a shortened version of the ACQ that omits the FEV1 measurement from the original ACQ score. The mean ACQ score is the average of the responses. A mean score ≤0.75 indicates well-controlled asthma, a score of 0.75 to ≤1.5 indicates partially controlled asthma, and a score >1.5 indicates poorly controlled asthma (Juniper et al, Respir Med. 100(4):616-21, 2006). An individual change of at least 0.5 is considered clinically significant (Juniper et al, Respir Med. 99(5):553-8, 2005). The Asthma Quality of Life Questionnaire, Standardized (AQLQ[S])+12 (AQLQ(S)+12) is a 32-item questionnaire that measures the HRQoL experienced by patients with asthma (Juniper et al, Chest. 115(5):1265-70, May 1999).An asthma diary will also be used for evaluation.

[0140] Treatment with anti-TSLP antibodies is effective in reducing asthma symptoms in aneosinophilic / hypeosinophilic populations as in hypereosinophilic populations, as disclosed in related U.S. Patent Publication No. 2018-0296669 (herein incorporated by reference). Methods of reducing the frequency of asthma exacerbations in a subject are also contemplated.

[0141] Methods of treating asthma in subjects with high or low Th2 asthma profiles are also contemplated herein. TSLP antagonists that inhibit the binding of the TSLP protein to its receptor complex are contemplated to effectively treat low eosinophilic asthma populations, as are the antibodies described herein. Similarly, TSLP antagonists that inhibit the binding of TSLP to its receptor complex are contemplated to be effective in treating low Th2 asthma populations. Methods for treating chronic obstructive pulmonary disease (COPD) in a subject are also contemplated, comprising administering an anti-TSLP antibody or antibody derivative or antigen binding protein described herein. It is contemplated that the subject being treated is a human. The subject may be an adult, an adolescent, or a child.

[0142] The therapeutic antibody (or antibody derivative) composition may be delivered to the patient at multiple sites. The multiple administrations may be administered simultaneously or over a period of time. In certain cases, it is beneficial to provide a continuous flow of the therapeutic composition. Additional therapy may be administered on a time-based basis, for example, hourly, daily, weekly, biweekly, triweekly, monthly, or at longer intervals.

[0143] In various embodiments, the amount of a therapeutic agent, such as a bivalent antibody having two TSLP binding sites, in a given dose can vary depending on the characteristics of the disorder being treated as well as the size of the individual to whom the treatment is being administered.

[0144] In an exemplary treatment, the anti-TSLP antibody or antibody derivative is administered in a dose range of about 70 mg to about 280 mg per daily dose. For example, doses may be given at about 70 mg, 210 mg, or 280 mg. In various embodiments, the anti-TSLP antibody or antibody derivative may be administered in a dose of 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 mg per dose. These concentrates may be administered as a single dosage form or as multiple doses. The above doses are given every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody derivative is administered in a single dose of 70 mg every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody derivative is administered in a single dose of 210 mg every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody derivative is administered in a single dose of 280 mg every two weeks or every four weeks.

[0145] In the case of antibody derivatives, the amount of antibody derivative should be such that the number of TSLP binding sites present in a dose has an equimolar number of TSLP binding sites as the canonical bivalent antibody described above.

[0146] It is contemplated that the anti-TSLP antibody or antibody derivative is administered every two weeks or every four weeks for a period of at least four months, six months, nine months, a year or more. In various embodiments, administration is subcutaneous or intravenous.

[0147] Treatment with an anti-TSLP antibody or antibody derivative is contemplated to reduce eosinophils in the blood, sputum, bronchoalveolar fluid, or lungs of a subject. It is also contemplated that administration will shift the subject's cell population from a high Th2 population to a low Th2 population. It is further contemplated that administration of an anti-TSLP antibody will improve one or more measures of asthma in a subject selected from the group consisting of forced expiratory volume (FEV), FEV1 reversibility, forced vital capacity (FVC), FeNO, Asthma Control Questionnaire-6 score, and AQLQ(S)+12 score.

[0148] Improvement in asthma may be measured as one or more of the following: reduction in AER (annualized exacerbation rate), reduction in asthma hospitalizations / severe exacerbations, change (increase) from baseline in time to first asthma exacerbation (after initiation of treatment with anti-TSLP antibody), reduction vs. placebo in the proportion of subjects with 1 or more asthma exacerbations or severe exacerbations over the course of treatment, e.g., 52 weeks, change (increase) from baseline in FEV1 and FVC (pre-bronchodilator and post-bronchodilator), change (decrease) from baseline in blood or sputum eosinophils (or lung eosinophils if biopsy or BAL fluid is available), change (decrease) from baseline in FeNO, change (decrease) from baseline in IgE, improvement in asthma symptoms and control as measured by PROs including ACQ and variants, AQLQ and variants, SGRQ, and asthma symptom diary, change (decrease) in use of rescue medication, reduction in use of systemic corticosteroids, reduction in blood Th2 / Th1 cell ratio. Most / all of these measurements should be in the whole population and subpopulations, including high and low eosinophils (high >250 and low <250), allergic and non-allergic, high and low Th2, high and low periostin (relative to the median), and high and low FeNO (>24 or <24).

[0149] Also contemplated in the present disclosure is the administration of multiple agents, such as an antibody composition in combination with a second agent as described herein, including, but not limited to, an anti-inflammatory agent or an asthma therapy.

[0150] However, in various embodiments, it is contemplated that this administration reduces the frequency or level of co-administered treatment in the subject.Exemplary co-administered treatments include, but are not limited to, inhaled corticosteroids (ICS), long-acting beta 2 agonists (LABA), leukotriene receptor antagonists [LTRA], long-acting antimuscarinics [LAMA], chromones, short-acting beta 2 agonists (SABA), and theophylline or oral corticosteroids.In various embodiments, this administration eliminates the need for corticosteroid treatment.

[0151] formulation In some embodiments, the present disclosure contemplates the use of pharmaceutical compositions comprising a therapeutically effective amount of an anti-TSLP antibody or antibody derivative together with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Additionally, the present disclosure provides methods of treating a subject by administering such a pharmaceutical composition.

[0152] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions may contain formulation materials to alter, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. These include, but are not limited to, ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18'' Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0153] Suitable vehicles or carriers may be water for injection, saline solution or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline, or saline mixed with serum albumin, are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, and may further comprise sorbitol or a suitable substitute thereof.

[0154] The formulation components are preferably present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically within a pH range of about 4.5 to about 8, such as about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0.

[0155] In various embodiments, the anti-TSLP antibody or antibody derivative is present in a formulation containing acetate and one or more of proline, sucrose, polysorbate 20, or polysorbate 80. In various embodiments, the formulation comprises 5-50 mM acetate, 3% (w / v) or less proline, 0.015% (w / v) ±0.005% (w / v) polysorbate 20 or polysorbate 80 at a pH of 4.9-6.0. Optionally, the antibody or antibody derivative is at a concentration of about 100 to about 150 mg / ml. The formulation may be stored at -20°C to -70°C. Exemplary anti-TSLP formulations containing these excipients are described in International Application No. PCT / US2021 / 018561, which is incorporated herein by reference.

[0156] In an alternative embodiment, the anti-TSLP antibody or antibody derivative is present in a formulation containing a surfactant and at least one basic amino acid or salt thereof. In a representative example, the basic amino acid is arginine or histidine. In various embodiments, the salt is arginine glutamate or histidine glutamate, optionally at a concentration of 10 to 200 mM. Optionally, the formulation further comprises proline. In an alternative embodiment, the anti-TSLP antibody or antibody derivative is present in a formulation containing a surfactant and calcium or a salt thereof. In various embodiments, the salt is calcium glutamate, optionally at a concentration of 15 mM to about 150 mM. Optionally, the formulation further comprises proline. In various embodiments, the surfactant is polysorbate 20 or polysorbate 80, or a mixture thereof. Optionally, the antibody or antibody derivative is present at a concentration of greater than about 110 mg / ml, or greater than about 140 mg / ml. Exemplary anti-TSLP formulations containing these excipients are described in International Patent Application No. PCT / US2021 / 017880, which is incorporated herein by reference.

[0157] When parenteral administration is contemplated, the therapeutic composition used may be provided in the form of a parenterally acceptable aqueous solution containing the desired anti-TSLP antibody or derivative thereof in a pyrogen-free, pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody is formulated as a sterile, isotonic solution, appropriately preserved. In certain embodiments, the formulation may include a formulation of the desired molecule with an agent that can provide a sustained or timed release of the product that can be delivered via depot injection, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid may also be used, which has the effect of enhancing duration in the circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the antibody. In various embodiments, administration may be via a prefilled syringe or an automatic injection device. In various embodiments, the automatic injection device is Ypsomed YpsoMate®. In various embodiments, the autoinjector is disclosed in WO 2018 / 226565, WO 2019 / 094138, WO 2019 / 178151, WO 20120 / 072577, WO 2020 / 081479, WO 2020 / 081480, PCT / US20 / 70590, PCT / US20 / 70591, PCT / US20 / 53180, PCT / US20 / 53179, PCT / US20 / 53178, or PCT / US20 / 53176.

[0158] kit In a further aspect, the present disclosure includes kits containing one or more compounds or compositions packaged in a manner that facilitates their use to practice the methods of the present disclosure. In one embodiment, such a kit includes a compound or composition described herein packaged in a container, such as a sealed bottle or container, with a label attached to the container or included within the package that describes the use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a particular route of administration or for performing a screening assay. Preferably, the kit includes a label that describes the use of the antibody composition.

[0159] Further aspects and details of the present disclosure will become apparent from the following examples, which are intended to be illustrative and not limiting. EXAMPLES

[0160] Example 1 - Identification of the attributes of Tezepelumab Tezepelumab is a full-length human monoclonal antibody of the IgG2 subclass produced in Chinese Hamster Ovary (CHO) cells. It is composed of two heavy chains (HC) and two light chains (LC) of the λ subclass. The heavy and light chains are covalently linked via disulfide bonds. Biochemical, biophysical, and biological characterization of tezepelumab was performed to provide a comprehensive understanding of its structural and functional properties and to enable evaluation of antibody attributes that may affect binding and efficacy.

[0161] Materials and Methods AMG157 and destabilizing residues that may affect binding: The amino acid sequence of AMG157 as sequence A5 (and chains H5, L5), as well as several other TSLP-binding antibodies, have been previously described in U.S. Pat. No. 7,982,016 B2.

[0162] The molecular weight of the A2G0F / A2G0F glycosylated antibody (C6500 H9998 O2068 N1734 S52) is 147189.4 Da, including removal of the N-terminal pyroglutamic acid and C-terminal K of the heavy chain. TSLP contained 74% monomeric species, 23% dimeric species, and 3% tetrameric species.

[0163] Peptide Mapping: Peptide mapping of tezepelumab samples was performed by a sample preparation procedure suitable for LC-MS analysis, including refolding with guanidine in peptides, reduction and alkylation of disulfide bonds, buffer exchange, and digestion with trypsin, as described in (Ren et al., Anal. Biochem. 392;12-21 (2009)). Briefly, samples containing tezepelumab were diluted to approximately 1 mg / ml in 0.5 ml of denaturing buffer pH 7.5 (7.5 M guanidine hydrochloride (GdnHCl) and 0.25 M Tris). Reduction was achieved by adding 3 μl of 0.5 M dithiothreitol (DTT) followed by incubation for 30 min at room temperature. Carboxymethylation was achieved by adding 7 μl of 0.5 M iodoacetic acid (IAA). The reaction was carried out for 15 min at room temperature in the dark. Excess IAA was quenched by adding 4 μl of 0.5 M DTT. The reduced and alkylated tezepelumab samples were buffer exchanged into digestion buffer (0.1 M Tris or 0.1 M ammonium bicarbonate) at pH 7.5 using a NAP-5 column (GE Healthcare, Piscataway, NJ, USA). Lyophilized trypsin was dissolved in water to a top concentration of 1 mg / ml. Digestion was initiated by adding 1 mg / ml trypsin solution to the reduced, alkylated, buffer exchanged tezepelumab samples to achieve an enzyme / substrate ratio of 1:25. Digestion was carried out for 30 min at 37°C. The final digest was quenched by adding 5 tl of 20% FA. LC-MS / MS peptide mapping analysis of digested tezepelumab samples was performed on an Agilent 1290 UHPLC system interfaced to a Thermo Scientific Q-Exactive Biopharma mass spectrometer as described in (Ren et al., 2009, supra). Using the resulting LC-MS / MS raw data and the sequences of tezepelumab and the target, modifications were identified and quantified by MassAnalyzer software (Zhang, Anal. Chem. 81;8354-8364(2009)).

[0164] SE-UHPLC: Tezepelumab samples were loaded onto an analytical SE-UHPLC column (BEH200 column, 1.7 μm particle size, 4.6 mm×150 mm, Waters Corporation) and proteins were separated isocratically using a mobile phase containing 100 mM sodium phosphate, 250 mM sodium chloride at pH 6.8. The eluent was monitored by ultraviolet absorbance at 280 nm. The column was operated at ambient temperature and the mobile phase was applied to the column at a flow rate of 0.4 mL / min.

[0165] Non-reduced RP-HPLC: Tezepelumab samples were analyzed by RP-HPLC using a Waters BEH300 C4 column (1.7 μm particle size, 2.1 mm×50 mm) and eluted with a mobile phase containing 0.1% TFA and a gradient of 1-propanol at 75° C. Absorbance at 215 nm was monitored.

[0166] Reduced CE-SDS: Tezepelumab samples were analyzed by rCE-SDS. Samples were reduced and denatured by heating in the presence of sodium dodecyl sulfate (SDS) and β-mercaptoethanol at pH 6.5, and then electroinjected into a bare fused silica capillary filled with SDS gel buffer at 25° C. The absorbance was monitored at 220 nm.

[0167] CEX-UHPLC: A sample of tezepelumab drug substance was loaded onto an analytical CEX-HPLC column (BioPro SP-F, 5 μm particle size, 4.6 mm × 100 mm, YMC America, Inc.). Mobile phase A contained 20 mM sodium phosphate at pH 6.6, and mobile phase B consisted of 20 mM sodium phosphate at pH 6.6, 500 mM sodium chloride. Proteins were separated using a linear salt gradient generated by 5% to 12% mobile phase B from 0 to 4 min, 23% mobile phase B at 18 min, 100% mobile phase B from 18.5 to 20.5 min, and back to 5% mobile phase B from 21 to 25 min. The eluate was monitored by ultraviolet absorbance at 280 nm. The column was operated at 28 °C, and the mobile phase was applied to the column at a flow rate of 0.6 mL / min.

[0168] Glycan Mapping: N-glycan mapping is an analytical technique that releases oligosaccharides linked to asparagine residues by enzymatic cleavage. The released oligosaccharides are then derivatized with fluorescent tags for detection and quantification. The labeled oligosaccharides are separated by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection to generate a glycan profile. In this method, tezepelumab is subjected to enzymatic digestion with N-glycosidase F (PNGase F), which specifically cleaves the bond between N-acetylglucosamine (GlcNAc) and asparagine residues in oligosaccharides. The released oligosaccharides are labeled with 2-aminobenzoic acid (2-AA) by reductive amination. After a centrifugation clean-up step, the oligosaccharides are separated by HILIC in an ultra-performance liquid chromatography (UPLC) system. The relative peak area percentages of the major oligosaccharide species are calculated.

[0169] Efficacy: The efficacy of tezepelumab compositions containing the attributes described herein was observed by receptor-ligand binding bioassays and / or reporter gene cell-based bioassays.

[0170] Receptor-Ligand Binding Assay: This assay provides a proximity measurement of tezepelumab activity and directly reflects the molecular mechanism of action of tezepelumab, which binds TSLP and prevents TSLP from binding to the TSLP receptor (TSLPR). This method provides a quantitative measurement of tezepelumab's ability to inhibit TSLP from binding to the TSLPR. Tezepelumab binds to recombinant TSLP-His ligand (TSLP-His) and inhibits TSLP-His from binding to the biotinylated TSLP receptor (TSLPR). The potency assay is a bead-based amplified luminescent proximity homogeneous assay ("Alpha") that detects biomolecular interactions. The assay includes two bead types, acceptor beads and donor beads. The donor beads are coated with a hydrogel containing phthalocyanine, photosensitizer, and streptavidin. The acceptor beads are coated with a hydrogel containing thioxene derivatives and nickel chelates. The donor bead binds to the biotinylated TSLPR through the interaction between streptavidin and biotin, and the acceptor bead binds to the histidine-tagged TSLP through the interaction between nickel chelate and histidine. When TSLP-His and biotinylated TSLPR bind to each other, the acceptor bead and the donor bead are brought into close proximity. When the complex is irradiated with a laser, the donor bead converts ambient oxygen to singlet oxygen. When the beads are in close proximity, energy transfer occurs to the acceptor bead, resulting in light emission, which is measured with a plate reader equipped with AlphaScreen® signal detection. Tezepelumab binds to TSLP-His and prevents TSLP-His from binding to biotinylated TSLPR, thereby reducing the light output in a dose-dependent manner. The activity of the test sample is measured by comparing the response of the test sample to the response obtained with the reference standard. It will be understood that the receptor-ligand binding assay described in this paragraph is a suitable assay for measuring the ability of a composition to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads.

[0171] Cell-based reporter gene bioassay: Human thymic stromal lymphopoietin (TSLP) protein binds to the human TSLP receptor (TSLPR) complex expressed on the surface of stable mouse BaF cells, inducing Stat 5 activation and cell proliferation. The method utilizes a mouse BaF / hu HTR cell line co-transfected with a plasmid encoding a Stat luciferase reporter gene and a blasticidin resistance gene. Incubation of Stat / BaF / HTR cells with recombinant human TSLP results in signal transduction after binding to the TSLPR, resulting in increased luciferase activity. AMG157 antagonizes the TSLP-induced activity of TSLPR, thereby inhibiting the luciferase reaction mediated by TSLP. The method measures the dose-dependent inhibitory effect of AMG 157 reference standard and test samples in TSLP-stimulated Stat / BaF / HTR cells. After incubation with TSLP and tezepelumab, the cells are treated with a reagent containing detergent (for cell lysis) and luciferin, a substrate for luciferase. The reaction of luciferase with luciferin produces luminescence, which is measured by a luminometer. The production of luciferase in reporter cells in response to stimulation with TSLP is quantified by reading the luminescence after adding a luciferase substrate. The degree of inhibition of TSLP-induced activity of luciferase reporter activity is proportional to the amount of tezepelumab. The biological activity of the test sample is measured by comparing the response of the test sample to a reference standard. It will be understood that the cell-based reporter gene assay described in this paragraph is a suitable assay for determining the ability of a composition to inhibit the binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, the expression of which indicates that TSLP binds to TSLPR.

[0172] result Biochemical characterization of tezepelumab has identified modified tezepelumab antibodies that can be isolated following storage of the tezepelumab drug product and drug substance, including isomerized, deamidated, oxidized, high molecular weight, fragmented, partially reduced, high mannose glycan, or disulfide isoform derivatives. These attributes were evaluated for their potential impact on the efficacy and tolerability of tezepelumab.

[0173] Isomerization: Aspartic acid isomerization was assessed by reduced peptide mapping by LC-MS / MS. Aspartic acid residues, whether native or formed by deamidation of Asn, can undergo isomerization via a cyclic imide intermediate. Isomerization can affect target binding and efficacy based on the proximity of the modified residue to the CDR. Native levels of tezepelumab isomerization were assessed by mass spectrometry in peptide mapping studies. HC CDR2 Asp 54 and LC CDR3 Asp 91 / 95 No significant level of isomerization at LC CDR2 Asp was observed in the drug substance. 49 / 50 It was shown that HC CDR2 Asp is prone to isomerization at high temperatures. 54 The isomerization level of LC CDR Asp also showed a slight increase (<2%) at high temperature. Thus, the predominant isomerization sites were LC CDR Asp at 10% and 2% after 5 weeks of thermally forced degradation (40 °C). 49 / 50 and HC CDR Asp 54 was identified as.

[0174] Light chain CDR2 Asp 49 / 50 Isomerization of HC CDR2 Asp was observed in approximately 0.2% of the drug substance. 54 and LC CDR3 Asp 91 / 95 Isomerization at 100% was not observed at significant levels in the drug substance.

[0175] Product lots used in clinical trials at the end of the shelf life were monitored for levels of impurities such as HMW species, fragments, isomerization, etc., and compared to impurities at the initial release of the same lot. The increase in impurities over time allows calculation of the subject's clinical exposure to the level of the impurity to determine the impact of these attributes on product safety, providing a measure of the tolerability of the impurities in the drug substance. For example, clinical trials of tezepelumab used drug substance administered in the final month of the clinical shelf life of up to 36 months. The use of aged formulations in combination with higher doses and more frequent dosing exposed patients to higher concentrations of product-related substances and product-related impurities than new formulation lots. The increased exposure was primarily due to the increased cumulative dose per month of treatment (e.g., with a 420 mg subcutaneous injection dose at Q14D) compared to a dose of 210 mg subcutaneous injection administered monthly.

[0176] The clinical dose of 420 mg subcutaneously every 2 weeks is approximately four times higher than the monthly dose of 210 mg. This dosing schedule resulted in a significant reduction in the "area under the curve" (AUC) or maximum serum concentration (C max The systemic exposure at the high-dose regimen, expressed as either the naïve or the naïve dose, was calculated to be 3.2- to 3.7-fold higher, respectively, than at the low clinical dose. According to the clinical trial protocol, antibody testing is performed only if there is an unexpected change in exposure or, in some cases, a safety event related to anti-drug antibodies. These outcomes were not observed and the drug was well tolerated.

[0177] Based on the doses and estimated attribute levels at doses in this study, attribute exposure levels for patients in the clinical study were estimated and tolerability was assessed. For example, the percentage of a drug product attribute (e.g., HWM) can be multiplied by a clinical exposure multiplier to determine the percentage of an equivalent attribute level in a production lot administered at the proposed dose of 210 mg at Q28D.

[0178] In the case of isomerization, a total amount of isomerization of up to 30% calculated based on a dose of 210 mg on Q28D was not associated with any in vivo safety issues. D49 / D50 or D52 isomerization of 26% calculated based on a dose of 210 mg on Q28D was not associated with any in vivo safety issues. Meanwhile, D54 isomerization of 4% calculated based on a dose of 210 mg on Q28D was not associated with any in vivo safety issues.

[0179] Oxidation: Oxidation was assessed using reduced tryptic peptide maps by LC-MS. Oxidation of methionine (Met) residues is a post-translational modification that can potentially occur as a result of exposure to oxygen and / or chemical oxidants, as well as light exposure. Tezepelumab contains eight Met residues in each heavy chain (Met 2 , Met 34 , Met 83 , Met 117 , Met 253 , Met 359 , Met 398 , Met 429 ). Met residues are absent in the light chain. The complementarity determining regions (CDRs) contain Met residues. 34 There is only one Met residue in the heavy chain, Met 2 , Met 117 , Met 253 , Met 359 , Met 398 Low but detectable levels of oxidation were observed in the methionine-containing CDRs (Table 1). Methionine oxidation within the CDRs could potentially affect potency, but the Met 34 No oxidation of tezepelumab was observed. The extent of oxidation was estimated by reduced peptide map with mass spectrometry detection (ESI-MS). Relative percentages were calculated by inference from the relative intensities of oxidized and non-oxidized species, but this approach is considered semiquantitative due to potentially different ionization efficiencies and co-elution of interfering species. Analysis of tezepelumab under forced oxidation revealed the susceptibility of specific sites on the molecule to oxidation.

[0180] [Table 1]

[0181] The order of sensitivity of heavy chain methionines to forced chemical oxidation is Met 117 >Met 253 >Met 2 >Met 429 It was shown that Met 117 and Met 253 Similar to the chemical oxidation studies described above, photolysis studies established the relative susceptibility of the heavy chain Met residues to light-induced oxidation as follows: Met 253 >Met 117 >Met 398 >Met 359 The level of oxidation of Met34 was below the quantitative level, suggesting that this residue is not available for oxidation, given the sequence and folding of the molecule. In addition, photolysis studies showed that Trp 102 >Trp 56 >Trp 52 >Trp 90 The oxidation level of tryptophan residues in the heavy chain variable region increased in the order of 102 and light chain Trp 56 was shown to be the site most exposed to light (Table 2).

[0182] [Table 2]

[0183] The oxidation observed at the end of shelf life (EOS) (maximum 36 months 2-8°C + 2 months 30°C) showed approximately 0.2% oxidation of W52 and 1.1% oxidation of W102 in the HCDR. For drug substance oxidation, 0.3-0.5% oxidized W102 was detected. The attribute exposure level for clinical trial patients was estimated based on the dose and estimated attribute level at administration in human clinical trials. The maximum 6-7% oxidized W102 calculated based on the 210 mg dose in Q28D was not associated with any in vivo safety issue. Tryptophan oxidation can occur under high temperatures and extreme visible and ultraviolet light exposure. Tryptophan oxidation in the CDR caused by extreme conditions is associated with a moderate reduction in potency. A strong correlation exists between tryptophan oxidation and the yellowness index.

[0184] Deamidation: Asparagine deamidation was assessed using tryptic peptide mapping by LC-MS. Native levels of deamidation of tezepelumab were assessed by ESI-MS / MS analysis of peptide mapping studies. 316 and Asn 385 Only low levels of deamidation were observed in the heavy chain CDR2 and light chain CDR1 (Table 3). 57 and Asn 25 / 26 Deamidation at other sites, including the , was not observed in the drug substance.

[0185] [Table 3]

[0186] The susceptibility of specific molecular sites to deamidation was assessed by analyzing tezepelumab under forced deamidation conditions. At physiological pH 7.4, the most sensitive site was Asn 390 and Asn 385 (3% in the drug substance, 5% in EOS), but the less sensitive site was Asn 316(0.09-0.1% in API and 0.4% in EOS), all of which are located within the Fc region. 25 Deamidation within the LC variable CDR region in was observed at low levels (0.1-0.2% in drug substance; 0.4% in EOS). Attributed exposure levels for clinical trial patients were estimated based on doses and estimated attributed levels at dosing in human clinical trials. A calculated maximum of 2% deamidation at Asn25 (based on a 210 mg dose in Q28D) was not associated with any in vivo safety concerns, and a calculated maximum of 13% deamidation at Asn385 / 390 (based on a 210 mg dose in Q28D) was not associated with any in vivo safety concerns.

[0187] Glycosylation: Glycosylation is involved in antibody effector functions and antibody binding to Fc receptors on cell surfaces, and mutations in glycosylation may disrupt one or more of these functions. Effector functions include antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

[0188] Tezepelumab was developed based on the presence of consensus sequences and the historical characterization of IgG2 monoclonal antibodies produced from mammalian cell culture, with Asn on each heavy chain. 298 It is predicted to contain a single N-glycosylation site at 1000 ng / ml. Glycosylation sites were assessed by comparing tryptic peptide maps with and without PNGaseF treatment. PNGaseF cleaves the mannose-rich hybrid complex glycan moiety between the N-acetylglucosamine residue at the reducing end of the glycan and the Asn residue in the peptide backbone. The species composition in the N-linked glycan map was confirmed by coupling the chromatographic separation outlet to an Orbitrap mass spectrometer.

[0189] A comprehensive characterization of the glycan complement of tezepelumab shows the presence of biantennary N-linked structures with various degrees of terminal galactosylation and high levels (approximately 95%) of fucosylation as the predominant species. The distribution of each glycan of the CEX drug substance as measured by HILIC is shown in Table 4.

[0190] [Table 4]

[0191] The tezeperumab glycan derivative population contains galactosylated species (19.9-28.6% in DS), afucosylated species (1.1-1.2% in DS), and 3.9-4.8% high mannose species (mainly oligomannose 5) in the DS. Attribute exposure levels for clinical trial patients were estimated based on doses and estimated attribute levels at dosing in human clinical trials. The calculated maximum of approximately 5% afucosylated species, calculated maximum of 75-90% galactosylated species, and calculated maximum of 14-18% high mannose derivatives are not associated with any in vivo safety concerns, and all estimates are based on a dose of 210 mg at Q28D.

[0192] To investigate the biological effect of N-linked glycan removal, tezepelumab was purified by treatment with PNGaseF and tested in receptor-ligand binding assays and cell-based reporter gene bioassays (Table 5). These results indicate that N-glycan removal does not affect tezepelumab in either potency assay.

[0193] [Table 5]

[0194] The level of high mannose glycans can potentially affect the half-life of the product, and the process conditions in the production bioreactor can affect the high mannose levels. When the effect of the production bioreactor process parameters on high mannose was evaluated in the process characterization studies, pH was identified as the main process parameter affecting high mannose. A pH tolerance range was established to support consistent high mannose levels. High mannose levels in the process characterization studies were ≤7.5%.

[0195] Example 2 - Size Derivatives In addition to chemical alterations at the amino acid level of tezepelumab, derivatives having aggregates or fragments are also available.

[0196] Assuming the presence of two unmodified light chains, two N-terminal pyroglutamidated heavy chains, and 18 disulfides, the predicted total mass of the intact tezepelumab peptide backbone is 144,298 Da. In addition, native tezepelumab has two Asn residues in each of the two heavy chains. 298 Tezepelumab contains a single N-linked glycosylation site at 1000 ng / ml. The theoretical mass of intact glycosylated tezepelumab with two copies of the glycan at Asn298, two copies of the predominant heavy chain C-terminal Gly derivative, and two copies of the predominant heavy chain N-terminal pyroglutamine is 147,189 Da. Treatment of intact tezepelumab with PNGaseF removes the N-glycosylation, with the glycan-bearing Asn being converted to an Asp residue, increasing the mass of the polypeptide by 1 Da per chain, and reducing the heterogeneity of the deconvoluted mass spectrum. The theoretical mass of the deglycosylated material is 144,300 Da.

[0197] Size heterogeneity is an inherent property of proteins due to the action of chemical or enzymatic cleavage as well as self-association by various mechanisms. Possible size derivatives may include high molecular weight (HMW) species that self-associate to form species larger than monomers (dimers, higher oligomeric species). HMW may be formed by non-covalent, reducible covalent, and / or non-reducible covalent bonds, and low molecular weight (LMW) species may be formed by truncation of the polypeptide backbone and / or incomplete association of subunit components (i.e., light and heavy chains).

[0198] The size heterogeneity of tezepelumab was assessed using the following analytical methods: size-exclusion ultra-performance liquid chromatography (SE-UHPLC) to assess size and purity under native conditions; SE-HPLC with sedimentation velocity ultracentrifugation (SV-AUC) and static light scattering (SLS) detection to provide additional molar mass estimates; reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) to measure size and purity under reduced denaturing conditions. Derivatives separated include fragments, non-reducing covalent bonds, polypeptides lacking normal glycosylation sites or containing additional glycosylation sites; non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS) to measure size and purity under denaturing conditions. Derivatives separated include partially assembled molecules, fragments, and covalent bonds.

[0199] Results from these analytical techniques showed that tezepelumab drug substance consisted primarily of monomers with low levels of dimers and LMW species based on the results of SE-UHPLC, SE-HPLC-SLS, and sedimentation velocity analytical ultracentrifugation (SV-AUC). Low levels of LMW species were observed under denaturing (nrCE-SDS) and reduced denaturing (rCE-SDS) conditions. Based on the rCE-SDS results, tezepelumab was primarily reduced to HC and LC components with minor levels of fragmentation and HMW species. These included LMW (smaller than LC), medium molecular weight (MMW, smaller than HC but larger than LC), and HMW (larger than HC) species. LMW species (e.g., <25 kD) and MMW species (approximately 25-50 kD) were collectively detected as fragments and were observed in less than 2% of tezepelumab formulations [DS: <0.4% (98.7-99% in HC+LC); EOS: 1.5% (97.3-97.5% in HC+LC)].

[0200] The size heterogeneity of tezepelumab was monitored by non-denaturing SE-UHPLC, which is an in-process control method and part of the drug substance and drug product release and stability testing program. The method is performed under non-denaturing conditions to separate the HMW species from the main monomer peak. Tezepelumab drug substance was analyzed by SE-UHPLC on a Waters BEH200 4.6×150 mm 1.7 mm particle size column in a mobile phase of 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8, at a flow rate of 0.4 mL / min, and absorbance at 280 nm was detected. The profile was determined by the presence of a main peak (relative area percentage 99.6%) that eluted at approximately 2.8 minutes. A minor peak was most commonly observed in the 20x concentrated chromatogram and elutes before the main peak with a retention time of approximately 2.2 minutes. This peak contains the HMW of tezepelumab and has a relative area percentage of 0.4% as shown in Table 6.

[0201] [Table 6]

[0202] Overall HMW species were found at approximately 0.3-0.6% in the drug substance (DS) but 1.7% in the EOS, e.g., ≤1.4% HMW(release) and ≤1.7% HMW(stability). Attribute exposure levels for clinical trial patients were estimated based on estimated attribute levels at dose and administration in human clinical trials. The calculated maximum HMW species of 20% HMW (based on a dose of 210 mg at Q28D) was not associated with any in vivo safety concerns.

[0203] rCE-SDS was used to evaluate the heavy and light chains, as well as the LMW and MMW species. The electropherogram of rCE-SDS of tezepelumab shows the peak area percentage values ​​shown in Table 7. These data indicate that tezepelumab is composed of disulfide-linked heavy and light chains. The minor peaks observed in the LMW and MMW regions of tezepelumab drug substance are within the baseline noise and variability of the method. Consistent with the SE-UHPLC results, few LMW or MMW species were observed. Based on the doses and estimated attribute levels at administration in human clinical trials, attribute exposure levels for clinical trial patients were estimated. The maximum of 15% fragment species calculated based on a dose of 210 mg at Q28D was not associated with any in vivo safety issues.

[0204] [Table 7]

[0205] CE-SDS can also be performed under non-reducing conditions to assess the presence of non-monomeric species. This technique is performed under denaturing conditions to unfold the protein and break non-covalent bonds, and is particularly useful for detecting partial molecular species and partially reduced intact molecules, i.e., molecules lacking the two light and two heavy chain components expected for a monomeric antibody, or one or more of the respective interchain bonds. Species consisting of two heavy chains associated with a single light chain (HHL), or a single heavy chain associated with a single light chain (HL, also known as a half molecule), have been reported from certain cell culture conditions (Trexler-Schmidt M, et al, 2010). Tezepelumab was denatured by heating in the presence of SDS and N-ethylmaleimide at pH 6.5, and then electroinjected into a bare fused silica capillary (50 mm ID x 30.2 cm) filled with SDS gel buffer at 25°C. The injection voltage was 10.0 kV, the separation voltage was 15.0 kV, and the absorbance was monitored at 220 nm. The data indicate that the tezepelumab drug substance consists primarily of disulfide-linked heavy and light chain monomers, with low levels of lower molecular weight species constituting less than 4.5% of the distribution (Table 8).

[0206] [Table 8]

[0207] The addition of static light scattering (SLS) detection to the SE-HPLC method allows for the determination of molar mass for individual peaks in the chromatogram. The intensity of light scattered by an eluting species is proportional to both the concentration and molecular weight of that species. The intensity of ultraviolet absorbance (280 nm) is proportional to the protein concentration. The molar mass of each eluting species can be determined by the instrument manufacturer's software using the light scattering intensity and concentration of each peak. Tezepelumab drug substance was analyzed by SE-HPLC chromatography coupled to online multi-angle light scattering detection using an Agilent 1100 HPLC system equipped with a TSK-GEL G3000SWxl, 5 μm particle size, 7.8 mm ID x 300 mm length column. The detectors used were a Wyatt Heleos II detector, a Wyatt Optilab TrEX RI detector, and an Agilent UV detector with a wavelength setting of 280 nm. The SE-HPLC runs were carried out at room temperature with 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8±0.1 buffer used as the mobile phase, and the flow rate was 0.5 mL / min.

[0208] The UV profile and corresponding molar mass calculated from the SLS data generated for tezepelumab drug substance show that the molar mass of the main peak is 145 kDa, which is in close agreement with the theoretical mass of the tezepelumab monomer (147 kDa). The peak eluting before the monomer has an average molar mass of 284 kDa, which is in close agreement with the theoretical mass of the tezepelumab dimer (294 kDa), indicating that the majority of the HMW species is tezepelumab dimer (Table 9).

[0209] [Table 9]

[0210] The enriched HMW fraction (enriched for tezepelumab dimer) and the main peak (containing mostly monomer) were evaluated for potency by receptor-ligand binding assays and cell-based reporter gene bioassays. Results show reduced potency with 64% and 62% tezepelumab activity measured by receptor-ligand binding assays and cell-based reporter gene bioassays, respectively (Table 10).

[0211] [Table 10]

[0212] This result predicts that self-association may force steric hindrance, leading to conformational changes that may in turn affect binding. Increased rates of aggregate formation may occur at elevated temperatures, low pH, physiological pH, and exposure to visible and ultraviolet light. Biological characterization confirmed that the HMW species exhibited reduced potency. In vitro potency reduction was only detectable when concentrated to levels significantly above those detected under normal processing and storage conditions.

[0213] Disulfide isoforms: Tezepelumab is an antibody of the IgG2 subclass and is therefore expected to exhibit disulfide-mediated structural derivatives and isoforms (Wypych et al., Journal of Biological Chemistry, Vol. 283(23):16194-16205, 2008; Dillon et al., Journal of Biological Chemistry, Vol. 283(23):16206-16215, 2008). Disulfide structural heterogeneity is inherent in recombinant and naturally occurring IgG2 molecules, which contain 18 disulfide bonds (6 interchain disulfide bonds and 12 intrachain disulfide bonds) (Wang et al, 2007; Zhang and Czupryn, 2002). The connectivity of disulfide bonds detected in tezepelumab was characterized using different techniques depending on the number of bonds present in the non-reduced peptide. For peptides containing a single disulfide bond, disulfide connectivity was determined by comparing reduced and non-reduced tryptic peptide maps.

[0214] Unlike the classical IgG2-A structure, the IgG2-B isoform contains two copies of the Fab peptide (C H 1-C L -hinge) with two copies of the hinge peptide. IgG2-A / B is represented by an intermediate form that combines some features of both IgG2-A and IgG2-B and is defined by an asymmetric arrangement that contains one Fab arm covalently linked to two copies of the hinge peptide via disulfide bonds (Wypych et al., Journal of Biological Chemistry, Vol. 283(23): 16194-16205, 2008; Dillon et al., Journal of Biological Chemistry, Vol. 283(23): 16206-16215, 2008; Zhang et al., Anal Chem., Vol. 82(3): 1090-1099, 2010).

[0215] Disulfide-bonded peptides were identified in the unfractionated drug substance by peptide mapping using the endoprotease trypsin under non-reducing and reducing conditions. The outlet of the RP-HPLC separation was coupled to an electrospray ionization mass spectrometer (ESI-MS) for mass spectrometry analysis in addition to UV detection. The non-reduced digest was subsequently treated with a reducing agent [tris(2-carboxyethyl)phosphine hydrochloride (TCEP)] and analyzed again using the same conditions. Each disulfide-bonded peptide from the non-reduced tryptic peptide map of the tezepelumab drug substance was analyzed for its constituent peptides by mass spectrometry analysis under reducing conditions. In summary, characterization of the disulfide-bonded peptides, designated A-H, revealed bonds between specific Cys residues, as summarized in Table 11, confirming the presence of the classical disulfide structure, IgG2-A.

[0216] [Table 11]

[0217] Non-reduced tryptic peptide maps also indicated the presence of IgG2-B derivatives. Furthermore, non-reduced RP-HPLC was used to confirm the presence of IgG2-B disulfide derivatives. In summary, characterization of disulfide-linked peptides A–D, F–G, and I revealed bonds between specific Cys residues, as summarized in Table 12, confirming the presence of the disulfide isoform structure of IgG2-B.

[0218] [Table 12]

[0219] The presence of structural isoforms, IgG2-A / B derivatives, was also found in the non-reduced tryptic peptide map of tezepelumab. Furthermore, the confirmation of IgG2-A / B disulfide isoform derivatives was performed by non-reduced RP-HPLC. In summary, characterization of disulfide-linked peptides A-G and peptide J revealed bonds between specific Cys residues, as summarized in Table 13, confirming the presence of disulfide isoform structures, IgG2-A / B.

[0220] [Table 13]

[0221] Comparison of reduced and non-reduced tryptic peptide mapping revealed not only disulfide-linked peptides predicted to be predominantly IgG2-A structure, but also peptides with additional connectivity associated with IgG2-A / B and IgG2-B disulfide structure isoforms. Relative levels of disulfide isoforms in tezepelumab ranged from approximately 3.4-4.2% IgG2-B, 39.2-42% IgG2-A / B, and 54.2-57.1% IgG2-A based on RP-HPLC. Attribute exposure levels for clinical trial patients were estimated based on doses and estimated attribute levels at dosing in human clinical trials. Up to 15% disulfide isoform derivatives of IgG2-B calculated based on a 210 mg dose in Q28D, and up to 75% disulfide isoform derivatives of IgG2-A / B calculated based on a 210 mg dose in Q28D, were not associated with any in vivo safety concerns. The potency of disulfide isoforms was assessed by enriching the major isoforms in the drug substance, IgG2-A, IgG2-A / B, and IgG2-B, using CEX-UHPLC, which demonstrated that all isoforms showed full potency within the assay performance.

[0222] Example 3 - Leverage Analysis of Attributes and Efficacy Statistical analysis was performed to assess the relationship between CDR isoAsp in the HMW species D49D50 and total CDR oxidation using a least squares regression model. The analysis was based on these attributes as determined from the forced degradation analysis described in Example 1.

[0223] Analyses were performed by potency measurements using the cell-based reporter gene bioassay described herein (Figure 1A-C) and by potency measurements using the receptor-ligand binding assay described herein (Figure 1D-F). The relationships between the identified attributes were comparable in both potency assays. A statistically significant negative correlation was identified between HMW species and total CDR trp oxidation (Figure 1B-C and 1E-F). The relationship between CDR IsoAsp D49D50 and potency did not reach statistical significance.

[0224] Example 4 - High Mannose Species and Pharmacokinetic (PK) Modeling A modeling approach was used to estimate the potential impact of increasing the high mannose (HM) fraction on the clearance of tezepelumab. The model assumed a half-life of 24.5 days (PK profile from intravenous administration of tezepelumab in clinical trials) and an increase rate constant of 0.035 for the HM form of tezepelumab on day 1, based on the rate of decrease in the HM fraction of a reference IgG2 monoclonal antibody after a single intravenous dose. The increase rate constant of the HM form of the reference IgG2 monoclonal antibody has the highest value analyzed to date and was selected as a conservative estimate of the half-life of the HM of tezepelumab. The PK profile was modeled to be a maximum of 122.5 days (equivalent to 5 half-lives) and no correction for selected pairings was used. The relationship between HM levels and the estimated increase in clearance of tezepelumab was modeled as shown in Table 14 below.

[0225] [Table 14]

[0226] These results show that tezepelumab compositions having 5% or less HM species showed little to no increase in clearance, and approximately 23% HM species in the tezepelumab composition resulted in approximately a 10% increase in antibody clearance.

[0227] All publications, patents, and patent applications described and cited herein are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodologies, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0228] Those skilled in the art will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise an isomerized derivative, and the amount of the isomerized derivative in the composition is less than about 30%, and the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and the tezepelumab is (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

2. The composition of claim 1, wherein the amount of said isomerized derivative in said composition is from about 0.5% to about 13%.

3. i) the isomerized derivative comprises a modification in a heavy or light chain complementarity determining region (CDR); ii) said isomerized derivative comprises a mutation at D54 in the heavy chain CDR of SEQ ID NO: 7, and / or at D49, D50 or D52 in the light chain CDR of SEQ ID NO: 4 in one or both of the variable region chains; iii) the isomerized derivative contains an isomerization at D54 of SEQ ID NO:7 in an amount of less than about 5%; iv) the isomerized derivative contains isomerization at one or more of D49, D50 or D52 of SEQ ID NO:4 in an amount of less than about 13%; and / or v) The composition according to claim 1 or 2, wherein the isomerized derivative is isoaspartic acid (isoAsp) or cyclic aspartic acid (cAsp).

4. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a deamidated derivative, and the amount of the deamidated derivative in the composition is less than about 15%, wherein the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and wherein the tezepelumab (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

5. i) the amount of said deamidated derivative in said composition is about 0.5% to 10%; ii) the deamidated derivatives comprise deamidated asparagine at N25 / N26 of SEQ ID NO:3, N316 of SEQ ID NO:13, and / or N385 / 390 of SEQ ID NO:13; iii) the deamidated derivative comprises deamidation at N25 / N26 of SEQ ID NO:3 in an amount of less than about 3%; and / or iv) The composition of claim 4, wherein the deamidated derivative comprises deamidation at one or more of N316 and / or N385 / 390 of SEQ ID NO: 13 in an amount of less than about 13%.

6. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise an oxidized derivative, and the amount of the oxidized derivative in the composition is less than about 7%, wherein the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and wherein the tezepelumab (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

7. i) the amount of said oxidized derivative in said composition is from about 0.4% to about 7%; ii) the oxidized derivative comprises an oxidation of one or more of heavy chain methionine M34 of SEQ ID NO:6, M253, M359 of SEQ ID NO:13, or heavy chain tryptophan W52 of SEQ ID NO:7, W90 of SEQ ID NO:5, or W102 of SEQ ID NO:8 in one or both of the heavy chains; iii) the oxidized derivative comprises an oxidation of one or more of the heavy chain methionines M34 of SEQ ID NO:6, M253, M359 of SEQ ID NO:13 in one or both of the heavy chains, optionally wherein the oxidation is in an amount of less than about 7%; and / or iv) The composition of claim 6, wherein said oxidized derivative comprises an oxidation of one or more of tryptophans W52 of SEQ ID NO:7, W90 of SEQ ID NO:5, or W102 of SEQ ID NO:8 in one or both of the heavy chains, optionally wherein said oxidation is in an amount of less than about 3%.

8. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives are high molecular weight (HMW) species, and the amount of the HMW species in the composition is less than about 20%, and wherein the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and wherein the tezepelumab (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

9. 9. The composition of claim 8, wherein the amount of HMW species in the composition is about 1.7% or less, or about 1.4% or less.

10. 10. The composition of claim 8 or 9, wherein the HMW species comprises a dimer of tezepelumab.

11. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a tezepelumab fragment, and the amount of the tezepelumab fragment in the composition is less than about 15%, and the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and the tezepelumab (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

12. The method of claim 1, wherein the tezepelumab fragment is a low molecular weight (LMW) or medium molecular weight (MMW) species, or a combination thereof; ii) the fragments are low molecular weight species less than about 25 kD; and / or iii) The composition of claim 11, wherein the fragments are medium molecular weight species having a molecular weight of about 25-50 kD.

13. 1. A composition comprising tezepelumab and one or more tezepelumab derivatives, wherein the one or more tezepelumab derivatives comprise a glycosylated derivative, and an amount of the glycosylated derivative in the composition is less than about 40%, and wherein the tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and wherein the tezepelumab (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

14. i) the amount of glycosylated derivatives in the composition is less than about 35%, about 30%, about 25%, about 20%, about 15%, about 10% or about 5%; ii) the glycosylated derivative comprises a glycosylation mutation of tezepelumab at residue N298 of SEQ ID NO: 13 in one or both heavy chains; and / or iii) the glycosylation derivative comprises afucosylation or a mutation of glycosylation of tezepelumab to a high mannose moiety or a galactosyl moiety.

15. The method of claim 1, wherein i) the glycosylated derivatives comprise afucosylated derivatives in an amount of less than about 5%; or ii) said glycosylated derivatives contain galactosyl moieties in an amount less than about 30%; iii) the glycosylated derivative contains high mannose moieties in an amount of less than about 5%; and / or iv) The composition of claim 13 or 14, wherein the tezepelumab and tezepelumab derivatives comprise about 25%, about 23%, about 21%, about 19%, about 17%, about 15%, about 13%, about 11%, about 8%, or about 5% or less of high mannose glycosylated derivatives.

16. 15. The composition according to claim 13 or 14, (a) the tezepelumab and tezepelumab derivatives have better efficacy and / or tolerability than compositions comprising more than 40% of the glycosylated derivatives, said efficacy comprising the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads, or the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, said expression of which indicates that TSLP binds to TSLPR; or (b) the tezepelumab and tezepelumab derivatives contain 15% or less high mannose and have lower clearance than compositions having greater than 15% high mannose; or (c) the tezepelumab and tezepelumab derivatives have better potency and / or tolerability than compositions comprising more than 40% of the glycosylated derivatives, said potency comprising the ability to inhibit the binding of biotinylated TSLPR immobilized on donor beads to TSLP-His immobilized on acceptor beads, or the ability to inhibit binding of TSLPR expressed on the surface of Stat / BaF / HTR cells encoding a Stat luciferase reporter gene, said expression of which indicates that TSLP binds to TSLPR; or (d) The tezepelumab and tezepelumab derivatives contain 25% or less high mannose and have lower clearance than compositions having more than 25% high mannose.

17. 1. A composition comprising tezepelumab and one or more disulfide isoform derivatives thereof, wherein tezepelumab and the one or more tezepelumab derivatives bind to a human thymic stromal lymphopoietin as set forth in SEQ ID NO:2, and the one or more disulfide isoform derivatives comprise an IgG2-B isoform and / or an IgG2-A / B isoform, and the amount of the disulfide isoform derivatives in the composition is less than about 75%.

18. The method of claim 1, wherein the one or more disulfide isoform derivatives comprises an IgG2-B isoform, and the amount of the disulfide derivatives in the composition is less than about 20% or less than about 5%; 20. The composition of claim 17, wherein ii) the one or more disulfide isoform derivatives comprises an IgG2-A / B isoform.

19. The method of claim 19, wherein i) the amount of the IgG2-A / B isoform in the composition is less than about 75%; or 20. The composition of claim 18, wherein ii) the amount of the IgG2-A / B isoform in the composition is from about 38% to about 43%.

20. A composition comprising tezepelumab that binds to human thymic stromal lymphopoietin as set forth in SEQ ID NO:2 and one or more tezepelumab derivatives, wherein the tezepelumab derivatives include isomerized derivatives, deamidated derivatives, oxidized derivatives, glycosylated derivatives, HMW species, fragments, disulfide isoform derivatives, or combinations thereof, wherein the composition comprises: (a) the amount of isomerized derivative in said composition is about 30% or less as measured by reduced peptide mapping; (b) the amount of deamidated derivatives in said composition is about 15% or less as measured by peptide mapping; (c) the amount of oxidized derivatives in said composition is about 7% or less as measured by reduced peptide mapping; (d) the amount of glycosylated derivatives in said composition is about 40% or less as determined by glycan mapping; (e) the amount of disulfide isoform derivatives in the composition is about 75% or less as measured by non-reducing reverse phase high performance liquid chromatography (RP-HPLC); (f) the amount of HMW species in the composition is about 20% or less, as measured by SE-HPLC; and / or (g) A composition having one or more of the following characteristics: the amount of fragments in said composition is about 15% or less as measured by rCE-SDS.

21. The composition according to any one of claims 17 to 20, wherein tezepelumab is (A) (i) the light chain CDR1 amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain variable domain comprising the light chain CDR3 amino acid sequence set forth in SEQ ID NO:5; and (B) (i) the heavy chain CDR1 amino acid sequence set forth in SEQ ID NO:6; (ii) the heavy chain CDR2 amino acid sequence set forth in SEQ ID NO:7; and (iii) a composition comprising a heavy chain variable domain comprising the heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:

8.

22. 21. The composition of any one of claims 1, 4, 6, 8, 11, 13, 17, and 20, wherein tezepelumab comprises a heavy chain amino acid sequence set forth in SEQ ID NO: 10 and a light chain amino acid sequence set forth in SEQ ID NO:

12.

23. 21. A pharmaceutical formulation comprising the composition of any one of claims 1, 4, 6, 8, 11, 13, 17, and 20, and one or more pharma- ceutically acceptable excipients.

24. 27. The composition of any one of claims 1, 4, 6, 8, 11, 13, 17, and 20 for use in treating an inflammatory disease in a subject.

25. 25. The composition of claim 24, wherein the inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic idiopathic urticaria, Ig-induced disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF).

26. The composition of claim 24, wherein the composition is administered at intervals of every two weeks or every four weeks.

27. The composition of claim 24, wherein the composition is administered for a period of at least 4 months, 6 months, 9 months, 1 year or longer.

28. The composition described in claim 25, wherein the asthma is severe asthma, eosinophilic asthma or non-eosinophilic asthma.

29. The composition of claim 24, wherein the administration is via a prefilled syringe or an automatic injection device.

30. The pharmaceutical formulation of claim 23 for use in treating an inflammatory disease in a subject.

31. The pharmaceutical formulation of claim 30, wherein the inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic idiopathic urticaria, Ig-induced disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF).

32. The pharmaceutical formulation of claim 30, wherein the pharmaceutical formulation is administered at intervals of every two weeks or every four weeks.

33. The pharmaceutical formulation of claim 30, wherein the pharmaceutical formulation is administered for a period of at least 4 months, 6 months, 9 months, 1 year or longer.

34. The pharmaceutical formulation of claim 31, wherein the asthma is severe asthma, eosinophilic asthma or non-eosinophilic asthma.

35. The pharmaceutical formulation of claim 30, wherein the administration is via a prefilled syringe or an automatic injection device.

36. 1. A method for assessing the quality of a tezepelumab composition, comprising: obtaining a tezepelumab composition comprising tezepelumab and one or more tezepelumab derivatives; measuring the amount of one or more tezepelumab derivatives in the composition, wherein the tezepelumab derivatives include isomerized derivatives, deamidated derivatives, oxidized derivatives, glycosylated derivatives, disulfide isoform derivatives, HMW species, fragments, or combinations thereof; comparing the measured amount of the one or more tezepelumab derivatives to a predetermined reference standard; and preparing a pharmaceutical formulation or pharmaceutical product of said tezepelumab composition if said comparison indicates that said predetermined reference standard is met.

37. The amount of isomerized derivatives is measured and said predetermined reference standard is about 30% or less; The amount of deamidated derivative is measured and said predetermined reference standard is about 15% or less; The amount of oxidized derivatives is measured and said predetermined reference standard is about 7% or less; The amount of glycosylated derivatives is measured and said predetermined reference standard is about 40% or less; the amount of disulfide isoform derivatives is measured and said predetermined reference standard is less than or equal to about 75%; The amount of HMW species is measured and said predetermined reference standard is about 20% or less; or 37. The method of claim 36, wherein the amount of fragments is measured and the predetermined reference standard is about 15% or less.

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