Antibodies against liraglutide and their use

Abstract

The present invention relates to particular antibodies and their uses, such as for identifying and / or quantifying liraglutide fibrils and / or semaglutide fibrils.

Description

Technical Field

[0001] The present invention relates to antibodies specific for fibrils of liraglutide or fibrils of semaglutide, and to the use of such antibodies.

[0002] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS-Web and incorporated herein by reference in its entirety. The ASCII copy created on March 30, 2020, is named 190042WO01 Sequence Listing_ST25.txt and is 111 kilobytes in size.

Background Art

[0003] Human GLP-1(7-37) and its analogs are known to tend to form various types of aggregates in solution. A particular type of such aggregate, referred to herein as fibrils, is irreversibly formed and is considered to be minimized in pharmaceuticals for administration to patients in liquid form. To date, the preferred method for assaying (i.e., identifying and / or quantifying) such fibrils has been based on thioflavin T (ThT), a fluorophore that changes the emission spectrum when it binds to fibrils. See, for example, Assay (V) herein. Assays for detecting peptide fibrils via ThT often involve first stressing the sample to amplify the amount of fibrils and enable detection, and the application of such stress is undesirable and time-consuming. There is also a desire for means to identify such peptide fibrils with higher sensitivity in mixtures containing the soluble form of the peptide.

Summary of the Invention

[0004] In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are prepared according to assay (I) herein. In some embodiments, the invention relates to an antibody that binds to semaglutide fibrils, which fibrils are prepared according to assay (II) herein. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a binding level to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, and the binding level is determined according to assay (III) at a liraglutide fibril concentration of at least 25 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower than the detection limit of liraglutide fibrils in the ThT assay, and the detection limit is determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a binding level to liraglutide fibrils that is at least 5-fold higher than the binding level of the antibody to soluble liraglutide, the antibody has a monomer purity of greater than 95%, and the binding level is determined according to assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower than the detection limit of liraglutide fibrils in the ThT assay, the antibody has a monomer purity of greater than 95%, and the detection limit is determined according to assay (VI-B) herein at a liraglutide fibril concentration of at least 0.025 μM.

[0005] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 115 and 121, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NO: 37, 38, and 39; SEQ ID NO: 43, 44, and 45; SEQ ID NO: 49, 50, and 51; SEQ ID NO: 55, 56, and 57; SEQ ID NO: 61, 62, and 63; SEQ ID NO: 67, 68, and 69; SEQ ID NO: 73, 74, and 75; SEQ ID NO: 79, 80, and 81; SEQ ID NO: 85, 86, and 87; SEQ ID NO: 91, 92, and 93; SEQ ID NO: 97, 98, and 99; SEQ ID NO: 103, 104, and 105; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NO: 115, 116, and 117; SEQ ID NO: 121, 122, 123; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 40, 41, and 42; SEQ ID NOs: 46, 47, and 48; SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 70, 71, and 72; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 100, 101, and 102; SEQ ID NOs: 106, 107, and 108; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 118, 119, and 120; SEQ ID NOs: 124, 125, and 126; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions.

[0006] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a variable region of the heavy chain as described in any one of the preceding embodiments and a variable region of the light chain as defined herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a sequence selected from the group consisting of SEQ ID NOs: 109 and 110, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 109 and 110.

[0007] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a variable light chain (VL) sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10.

[0008] In some embodiments, the present invention relates to the use of an antibody as defined herein for the identification of liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to a method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising a) binding an antibody as defined herein to liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to a method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising a) binding an antibody as defined herein to liraglutide fibrils or semaglutide fibrils.

[0009] In some embodiments, the present invention relates to the use of an antibody as defined herein for the purification of liraglutide or semaglutide by removal or reduction of liraglutide or semaglutide fibrils, by immobilizing the antibody on a solid surface, such as a chromatographic surface or a membrane surface that makes an affinity surface, and exposing a mixture comprising both fibrillar and soluble forms of liraglutide or semaglutide to the surface, resulting in the isolation of the fibrils or a portion thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

Figure 1

[0011] The present invention relates to antibodies that specifically bind to the fibrils of the GLP-1 receptor agonists liraglutide or semaglutide. Liraglutide and semaglutide are analogs of human GLP-1(7-37) and are therapeutic peptides commercially available in solution form. The fibrils of liraglutide or semaglutide are undesirable in pharmaceuticals. Thus, the antibodies of the present invention make it possible to distinguish the fibrils of liraglutide or semaglutide from their soluble forms. Such antibodies of the present invention optionally enable the identification and / or quantification of such fibrils in a mixture with their soluble forms, and have several technical advantages including providing a means for ensuring the adequate quality of pharmaceuticals containing liraglutide or semaglutide. In some embodiments, the antibodies of the present invention enable the isolation or partial isolation of liraglutide fibrils from a mixture of soluble liraglutide. Such isolation may be performed by immobilization on a solid surface, such as a chromatography column, filter, or membrane. In some embodiments, the antibodies of the present invention optionally enable a highly sensitive assay for detecting very low levels of peptide fibrils in the presence of a very large excess of the soluble form of the peptide. In some embodiments, with respect to the specific peptides liraglutide or semaglutide, the terms "fibril", "peptide fibril" refer to the type of aggregates that can be obtained according to assay (I) herein for liraglutide or according to assay (II) herein for semaglutide, and such fibrils appear, for example, in the shape of thin threads when using transmission electron microscopy.

[0012] The inventors have surprisingly discovered that the antibodies of the present invention are at least 100-fold, and perhaps even at least 1000-fold, more sensitive for detecting fibrils compared to ThT assays such as ThT assays without agitation, for example, assay (V) herein.

[0013] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are prepared according to assay (I) herein. In some embodiments, the present invention relates to an antibody that binds to semaglutide fibrils. In some embodiments, the present invention relates to an antibody that binds to semaglutide fibrils, wherein the fibrils are prepared according to assay (II) herein. In some embodiments, the antibody has a detection limit for liraglutide fibrils at a concentration at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower, than the detection limit of liraglutide fibrils in a ThT assay, which detection limit is optionally determined according to assay (VI) herein. In some embodiments, the antibody has a binding level to liraglutide fibrils at least 10-fold higher, such as at least 20-fold higher or at least 50-fold higher, than the binding level of the antibody to soluble liraglutide. In some embodiments, the antibody has a detection limit for semaglutide fibrils at a concentration at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower, than the detection limit of semaglutide fibrils in a ThT assay, which detection limit is optionally determined according to assay (VI) herein. In some embodiments, the antibody has a binding level to semaglutide fibrils at least 10-fold higher, such as at least 20-fold higher or at least 50-fold higher, than the binding level of the antibody to soluble semaglutide. In some embodiments, the binding level is determined according to assay (IV) herein. In some embodiments, the binding level is determined according to assay (III) herein. In some embodiments, the binding level is determined according to assay (III-B) herein.

[0014] In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a binding level to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, and the binding level is determined according to assay (III) herein at a liraglutide fibril concentration of at least 25 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower than the detection limit of liraglutide fibrils in the ThT assay, and the detection limit is determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a binding level to liraglutide fibrils that is at least 5-fold higher than the binding level of the antibody to soluble liraglutide, the antibody has a monomer purity of greater than 95%, and the binding level is determined according to assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, which fibrils are optionally prepared according to assay (I) herein, and the antibody has a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower than the detection limit of liraglutide fibrils in the ThT assay, the antibody has a monomer purity of greater than 95%, and the detection limit is determined according to assay (VI-B) herein at a liraglutide fibril concentration of at least 0.025 μM.

[0015] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, and the antibody can detect liraglutide fibrils at a concentration of 1 to 1000 ppm of fibrils in solution, such as 1 to 10 ppm of fibrils, or 10 to 100 ppm of fibrils, or 100 to 1000 ppm of fibrils.

[0016] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 115 and 121, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 37, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 43, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 49, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 55, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 61, or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 67 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 73 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 79 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 85 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 91 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 97 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 103 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 115 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises the CDR3 sequence of SEQ ID NO: 121 or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.

[0017] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 37, 38, and 39; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 55, 56, and 57; SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 79, 80, and 81; SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 91, 92, and 93; SEQ ID NOs: 97, 98, and 99; SEQ ID NOs: 103, 104, and 105; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 115, 116, and 117; SEQ ID NOs: 121, 122, and 123; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 37, 38, and 39; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 55, 56, and 57; SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 67, 68, and 69; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 79, 80, and 81; SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 91, 92, and 93; SEQ ID NOs: 97, 98, and 99; SEQ ID NOs: 103, 104, and 105; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 115, 116, and 117; SEQ ID NOs: 121, 122, 123; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 37, 38, and 39; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 43, 44, and 45; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 49, 50, and 51; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 55, 56, and 57; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 61, 62, and 63; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 67, 68, and 69; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 73, 74, and 75; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 79, 80, and 81; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 85, 86, and 87; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 91, 92, and 93; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 97, 98, and 99; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 103, 104, and 105; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 115, 116, and 117; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the variable region of the heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 121, 122, and 123; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.

[0018] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 40, 41, and 42; SEQ ID NOs: 46, 47, and 48; SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 70, 71, and 72; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 94, 95, and 96; SEQ ID NOs: 100, 101, and 102; SEQ ID NOs: 106, 107, and 108; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 118, 119, and 120; SEQ ID NOs: 124, 125, and 126; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 40, 41, and 42; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 46, 47, and 48; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 52, 53, and 54; or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 58, 59, and 60; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 64, 65, and 66; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 70, 71, and 72; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 76, 77, and 78; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 82, 83, and 84; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 88, 89, and 90; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 94, 95, and 96; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 100, 101, and 102; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 106, 107, and 108; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 118, 119, and 120; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of SEQ ID NOs: 124, 125, and 126; or any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody comprises a variable region of the heavy chain as defined herein and a variable region of the light chain as described in any one of the preceding embodiments.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a sequence selected from the group consisting of SEQ ID NOs: 109 and 110, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 109 and 110. In some embodiments, the antibody has at least 70% sequence identity, such as at least 75%, to a sequence as defined herein. In some embodiments, the antibody has at least 80% sequence identity, such as at least 85% or at least 90%, to a sequence as defined herein. In some embodiments, the antibody has at least 91% sequence identity, such as at least 92% or at least 93%, to a sequence as defined herein. In some embodiments, the antibody has at least 94% sequence identity, such as at least 95% or at least 96%, to a sequence as defined herein. In some embodiments, the antibody has at least 97% sequence identity, such as at least 98% or at least 99%, to a sequence as defined herein.

[0019] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a variable light chain (VL) sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10.

[0020] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a single-chain Fv fragment. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the antibody is a single-chain Fv fragment further comprising an Fc domain. In some embodiments, the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. In some embodiments, the antibody specifically binds to the liraglutide fibrils. In some embodiments, the antibody specifically binds to the semaglutide fibrils.

[0021] In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 70%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 75%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 80%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 85%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 90%. In some embodiments, the antibody that binds to liraglutide fibrils has a monomer purity of greater than 95%. In some embodiments, the purity of the antibody that binds to liraglutide fibrils is determined according to the method described herein in "size exclusion chromatography", followed by the area under the curve (AUC 280nm based on the absorbance at 280 nm of the peak of the monomer antibody associated with the sum of the AUCs of all peaks 280nm ).

[0022] Liraglutide and Semaglutide Liraglutide and semaglutide are analogs of human GLP-1(7-37) containing a covalent moiety. The antibodies of the present invention bind to liraglutide fibrils and / or semaglutide fibrils. The term "fibril" as used herein with respect to liraglutide refers to liraglutide fibrils, and as used herein with respect to semaglutide refers to semaglutide fibrils. In some embodiments, the antibody of the present invention binds to liraglutide fibrils. In some embodiments, the antibody of the present invention binds to semaglutide fibrils.

[0023] Liraglutide is Arg34,Lys26-(N-epsilon-(gamma-L-glutamyl(N-alpha-hexadecanoyl)))-GLP-1(7-37) and can be prepared according to Example 37 of WO98 / 08871, which is incorporated herein by reference. The structure of liraglutide is also published in WHO Drug Information Vol.17, No.2, 2003. The structure of liraglutide is also published in WHO Drug Information Vol.24, No.1, 2010. Liraglutide fibrils can be prepared as described in Assay (I) herein. An example of soluble liraglutide is a commercially available solution manufactured by Novo Nordisk A / S, Denmark, for example, the trademark Victoza®.

[0024] Semaglutide is N-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37) peptide and can be prepared according to Example 4 of WO2006 / 097537, which is incorporated herein by reference. Semaglutide fibrils can be prepared as described in Assay (II) herein. Examples of soluble semaglutide are commercially available solutions manufactured by Novo Nordisk A / S, Denmark, for example, the trademark Ozempic®.

[0025] antibody In some embodiments, the invention relates to one or more of a series of antibodies characterized by antibody functionality and / or the amino acid sequence of a CDR, variable region of the heavy chain, variable region of the light chain, and / or sequence of the Fc domain. In some embodiments, as used herein, the term "CDR" is determined according to the Kabat antibody numbering scheme (Kabat, Elvin A. (1976). Structural Concepts in Immunology and Immunochemistry. New York, NY, USA: Holt, Rinehart & Winston). In some embodiments, the invention relates to one or more of a series of antibodies characterized by antibody functionality and / or the H-CDR3 amino acid sequence. In some embodiments, the invention relates to one or more of a series of antibodies characterized by antibody functionality and / or the CDR amino acid sequence (the CDR1, CDR2, and CDR3 of the variable region of the heavy chain may be referred to herein as H-CDR1, H-CDR2, and H-CDR3. Similarly, the CDR1, CDR2, and CDR3 of the variable region of the light chain may be referred to herein as L-CDR1, L-CDR2, and L-CDR3). In some embodiments, the invention relates to one or more of a series of antibodies characterized by antibody functionality and / or the amino acid sequences of the variable regions of the heavy and light chains. In some embodiments, the invention relates to one or more of a series of antibodies characterized by antibody functionality and / or the amino acid sequences of the variable regions of the heavy and light chains, and / or the sequence of the Fc domain. In some embodiments, the antibody comprises an H-CDR3. In some embodiments, the antibody comprises H-CDR1, H-CDR2, and / or H-CDR3. In some embodiments, the antibody comprises H-CDR1, H-CDR2, and H-CDR3. In some embodiments, the antibody comprises L-CDR1, L-CDR2, and / or L-CDR3. In some embodiments, the antibody comprises L-CDR1, L-CDR2, and L-CDR3. In some embodiments, the antibody comprises the variable region of the heavy chain and / or the variable region of the light chain.

[0026] The antibodies of the present invention may be in any form, including whole antibodies and antigen-binding fragments (i.e., "antigen-binding portions") or single-chain antibodies.

[0027] In some embodiments, the antibody is a single-chain variable fragment (scFv) antibody. In some embodiments, the antibody is a single-chain variable fragment fused to an Fc domain (scFv-Fc) antibody. In some embodiments, the scFv or scFv-Fc antibody consists of one amino acid sequence containing the variable region of the heavy chain (V H ) and the variable region of the light chain (V L ), and the scFv-Fc antibody further contains an Fc domain.

[0028] In some embodiments, the antibody is a full-length antibody, for example, containing standard antibody domains and regions as described herein. A full-length antibody (or whole antibody) contains four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a variable region of the heavy chain (V H ) and a constant region of the heavy chain (C H ). Each light chain contains a variable region of the light chain (V L ) and a constant region of the light chain (C L ). The constant region of the heavy chain contains three domains, C H 1, C H 2, and C H 3. Each light chain contains a variable region of the light chain (abbreviated as V L herein) and a constant region of the light chain. The constant region of the light chain contains one domain, C L .

[0029] The variable region of the heavy chain and the variable region of the light chain each contain a binding domain that interacts with the antigen. The V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V Lmay include three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0030] In some embodiments, the antibody is an antibody fragment, and such fragments can be obtained using conventional recombinant or protein engineering techniques. The antibody fragments of the invention can be made by truncation, for example, by removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Also, the fragments can be generated by one or more internal deletions. In some embodiments, the antibodies of the invention are a fragment of, or include, any one of the antibodies described herein. In some embodiments, the antibodies of the invention are an antigen-binding portion of, or include, one of the antibodies described herein, or a variant thereof. For example, the antibodies of the invention can be a Fab fragment of, or a variant thereof, one of the antibodies described herein, or the antibodies of the invention can be a single-chain antibody, or a variant thereof, derived from one of the antibodies described herein. Examples of antigen-binding fragments include Fab, Fab’, F(ab)2, F(ab’)2, Fv (typically, the V L and V H ) of a single arm of the antibody), single-chain Fv (scFv; see, e.g., Bird et al., Science 1988;242:42S-426, and Huston et al. PNAS 1988;85:5879-5883), Fd (typically, V H and C H 1), and dAb (typically, V H ) fragments; V H , V L , VhH, and V-NAR; single V H and single V LMonovalent molecules comprising a lock; minibodies, diabodies, triabodies, tetra-bodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camelid IgG; IgNAR; and one or more isolated CDRs or functional paratopes, wherein the isolated CDRs or antigen-binding residues or polypeptides can associate or link together to form a functional antibody fragment. Various types of antibody fragments are described or contemplated, e.g., in Holliger and Hudson, Nat Biotechnol 2005; 23:1126-1136, WO2005 / 040219, and U.S. Patent Application Publication Nos. 2005 / 0238646 and 2002 / 0161201.

[0031] The terms "complementary determining region" ("CDR") or "hypervariable region", as used herein, refer to the amino acid residues of an antibody that are involved in antigen binding. CDRs generally consist of CDR1, CDR2, and CDR3 within the variable region of the light chain and CDR1, CDR2, and CDR3 within the variable region of the heavy chain, as defined generally according to Kabat, and / or residues from "hypervariable loops" (Chothia and Lesk, J. Mol. Biol 1987; 196: 901-917). Typically, the numbering of amino acid residues within this region is carried out by the method described by Kabat et al. (supra). As used herein, the term "Kabat" refers to, for example, the numbering system for the variable region of the heavy chain and / or the variable region of the light chain described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to deletions or insertions into the framework (FR) or CDRs of the variable region. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology between the antibody's sequence and the "standard" Kabat numbering sequence. The term "framework region" or "FR" residues, as defined herein, refers to these V H or V L amino acid residues that are not within the CDRs. The fragment crystallizable region (Fc domain) of an antibody is the region of the antibody that can interact with cell surface receptors called Fc receptors, as well as with some proteins of the complement system.

[0032] The term "antibody derivative" refers to any modified form of an antibody, such as an antibody conjugated to another agent or conjugate of antibodies.

[0033] The term "antigen" can refer to a molecular entity used to generate an antibody. However, as used herein, the term "antigen" broadly refers to a target molecule that binds or specifically binds to an antibody, and thus includes fragments or mimetics of a molecular entity used to generate an antibody. Antibodies can be generated by any method including immunization of an animal or by display screening, such as phage display or yeast display.

[0034] As used herein, the term "epitope" is defined in the context of the molecular interaction between an "antigen-binding polypeptide", such as an antibody or a fragment thereof, and its corresponding antigen. Generally, an "epitope" refers to the area or region on an antigen to which an antibody binds or specifically binds, i.e., the area or region that is in physical contact with the antibody. An epitope can include amino acid residues in the antigen that are directly involved in binding to the antibody (also referred to as the immunodominant component of the epitope), as well as other amino acid residues that are not directly involved in binding, such as amino acid residues of the antigen that are effectively blocked by the antibody (in other words, these amino acid residues are within the "solvent-excluded surface" and / or "footprint" of the antibody). A given antigen may contain a number of different epitopes, including linear peptide epitopes, conformational epitopes consisting of one or more non-contiguous amino acids that are in close proximity to each other in the native (mature) conformation, and post-translational epitopes consisting of all or part of a molecular structure covalently attached to the antigen, such as a carbohydrate group, but are not limited thereto.

[0035] The terms "binding", "specific binding", and "specificity" of an antibody are used herein to describe the selectivity of the antibody or its antigen-binding fragment. The antibodies according to the invention can specifically bind to liraglutide fibrils or semaglutide fibrils, indicating that the antibodies have significantly lower binding levels to other antigens. In some embodiments, significantly lower means at least 10-fold lower binding levels, such as at least 15-fold lower or at least 20-fold lower. The binding levels can be determined according to Assay (III) herein or according to Assay (IV) herein. The binding levels can be determined according to Assay (III-B) herein.

[0036] As used herein, the term "sequence identity" refers to the degree of relatedness between polypeptide sequences determined by the number of matches between two or more chains of amino acid residues, and can be determined as the percentage of exact matches between the smaller of two or more sequences having a gap alignment (if any) processed by a particular mathematical model or computer program (i.e., an "algorithm"). The sequence identity of a polypeptide can be readily calculated by methods known in the art, including but not limited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining sequence identity are designed to give the maximum match between the sequences being tested.Methods for determining sequence identity are described in published computer programs, and such preferred computer program methods for determining sequence identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is published by the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm can also be used to determine sequence identity. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "match span" determined by the algorithm). A gap start penalty (calculated as three times the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used, and the "diagonal" is the score or numerical value assigned to each perfect amino acid match by a particular comparison matrix), and a gap extension penalty (usually {fraction (1 / 10)} times the gap start penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm.Standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978); for the BLOSUM 62 comparison matrix, see Henikoff et al., Proc. Natl. Acad. Sci USA 89, 10915-10919 (1992)) are also used by the algorithm. In some embodiments, sequence identity is determined using the following parameters, for example, with the algorithm GAP: Algorithm: Needleman et al., J. Mol. Biol. 48, 443-453 (1970), comparison matrix: BLOSUM 62 from Henikoff et al., PNAS USA 89, 10915-10919 (1992), gap penalty: 12, gap length penalty: 4, similarity threshold: 0, and no penalty for end gaps.

[0037] In some embodiments, the antibodies of the invention include one or more amino acid substitutions or insertions. The amino acid substitutions may be in the form of conservative amino acid substitutions. A "conservative amino acid substitution" may involve substituting one amino acid residue with another such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions may be made within the following groups of amino acids: hydrophilic: Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; aliphatic: Val, Ile, Leu, Met; basic: Lys, Arg, His; aromatic: Phe, Tyr, Trp; and further, typically, any residue may be substituted with alanine.

[0038] In some embodiments, one or more non-natural amino acids are introduced by substitution or insertion into the antibodies of the invention. Such non-natural amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as Cα-methyl amino acids, and Nα-methyl amino acids.

[0039] Amino acid sequence variants of the antibody of the present invention can be prepared by introducing appropriate nucleotide changes into the nucleic acid of the present invention or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Combinations of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final polypeptide product has the desired characteristics. Variant (modified) polypeptides can be prepared using any technique known in the art. For example, the polynucleotide of the present invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as E. coli XL-I red (Stratagene), and growing the transformed bacteria for a suitable number of generations. Products derived from the mutated / modified DNA can be easily screened using the techniques described herein to determine whether they have receptor binding and / or inhibitory activity. In designing amino acid sequence variants, the location of the mutation site and the nature of the mutation depend on the feature being modified. The mutation site can be modified individually or sequentially, for example, (1) by first substituting with conservative amino acid selections and then with more radical selections depending on the results achieved, (2) by deleting the target residue, or (3) by inserting other residues adjacent to the site where they are placed. In some embodiments, the amino acid sequence deletion extends over about 1 to 15 residues, more preferably about 1 to 10 residues, and typically about 1 to 5 consecutive residues.

[0040] In some embodiments, the molecule consists essentially of a defined sequence. In some embodiments, the molecule consists of a defined sequence. In some embodiments, the antibody is an isolated antibody. The term "isolated antibody" refers to an antibody that has been separated and / or recovered from another / other components in its natural environment and / or purified from a mixture of components in its natural environment. The antibodies of the present invention may be from different species, including mammalian species such as mouse, rat, rabbit, pig, or non-human primate. The antibody may be a rodent antibody, more specifically a mouse antibody. Alternatively, the antibody may be from a non-mammalian species such as chicken. The antibody may further be a humanized antibody or a human antibody.

[0041] The antibodies of the present invention can be prepared according to methods known in the art such as recombinant proteins, cell culture, and immunological techniques. Such techniques are described and explained throughout the source literature such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley and Sons (including all revisions to date).

[0042] Single-chain antibodies, including scFv or scFv-Fc antibodies, can be prepared by inserting a DNA sequence corresponding to their amino acid sequence into a plasmid in a host cell and then expressing the antibody using this host cell by recombinant techniques, such as bacterial cell culture, and such methods are well known in the art.

[0043] Monoclonal antibodies are typically produced by fusing myeloma cells with spleen cells from a mouse immunized with the desired antigen. Human monoclonal antibodies can be obtained from transgenic animals (e.g., mice or other suitable species) encoding human antibodies. Alternatively, recombinant monoclonal antibodies can be produced with techniques referred to as repertoire cloning or phage display / yeast display. Recombinant antibody engineering involves producing antibodies using viruses or yeast rather than mice.

[0044] Methods and Uses of Antibodies In some embodiments, the present invention relates to the use of an antibody as defined herein for identifying and / or quantifying liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to the use of an antibody as defined herein for isolating, including partially isolating liraglutide fibrils or semaglutide fibrils from a solution containing soluble liraglutide or soluble semaglutide. Such identification and / or quantification can be performed, for example, via an enzyme-linked immunosorbent assay (ELISA) by binding the antibody to the fibrils and subsequently detecting the bound antibody. The ELISA can be performed as known in the art. In some embodiments, the container for the ELISA (such as a microtiter plate) is first saturated. The saturation can involve a protein such as lysozyme or albumin, e.g., bovine serum albumin (BSA) or ovalbumin. In some embodiments, the antibody of the present invention bound to the fibrils is bound to a secondary antibody. If the antibody of the present invention contains an Fc domain, the secondary antibody can bind to this Fc domain. If there is a marker on the secondary antibody, detection and / or quantification of the secondary antibody may be possible, and such a marker can be a fluorophore that can be identified via spectroscopy. Quantification can be performed using a standard of the fibrils bound to the antibody of the present invention.

[0045] In some embodiments, as used herein, the term "detection limit" refers to the lowest limit of detection, which is the lowest concentration of a substance at which the presence of the substance can be distinguished from its absence. In some embodiments, as used herein, the term "detection limit" refers to the 3 mixture / soluble specificity ratio determined according to Assay (IV) herein. A comparison of the detection limits using the antibody and ThT assays can be performed according to Assay (VI) herein. A comparison of the detection limits using the antibody and ThT assays can be performed according to Assay (VI-B) herein. In some embodiments, as used herein with respect to an antibody, the term "detection limit" refers to the detection limit of an assay using the antibody in ELISA, such as Assay (III) or Assay (IV) herein. In some embodiments, as used herein with respect to an antibody, the term "detection limit" refers to the detection limit of an assay using the antibody in ELISA, such as Assay (III-B) herein. In some embodiments, as used herein, the term "detection limit" is three times the standard deviation of control samples tested in duplicate, and the standard deviation can be determined by Student's t-test.

[0046] In some embodiments, the invention relates to the use of an antibody as defined herein for the identification of liraglutide fibrils or semaglutide fibrils.

[0047] In some embodiments, the invention relates to the use of an antibody as defined herein as an affinity ligand for removing fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide, or (ii) semaglutide fibrils and soluble semaglutide.

[0048] In some embodiments, the invention relates to a method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising a) binding an antibody as defined herein to the liraglutide fibrils or semaglutide fibrils.

[0049] In some embodiments, the present invention relates to a method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising: a) binding an antibody as defined herein to the liraglutide fibrils or semaglutide fibrils; b) further comprising detecting the antibody bound to the liraglutide fibrils or semaglutide fibrils, the method according to any one of the preceding embodiments. In some embodiments, the method further comprises: c) optionally quantifying the antibody bound to the liraglutide fibrils or semaglutide fibrils by use of a standard of the fibrils. In some embodiments, the fibrils are in solution. In some embodiments, the fibrils are in a solution further comprising soluble liraglutide. In some embodiments, the fibrils are in a solution that further does not contain other peptides or proteins other than liraglutide fibrils and optionally soluble liraglutide.

[0050] In some embodiments, the method comprises: (a) contacting a solid support with a sample under conditions such that one or more fibrils in the sample are immobilized on the solid support; (b) contacting the solid support with any one of the antibodies described herein or an antigen-binding fragment thereof under conditions such that the antibody binds to the one or more immobilized fibrils to form an antibody-fibril complex; (c) contacting the antibody-fibril complex with a second antibody comprising a detectable label, wherein (i) the second antibody specifically binds to the antibody-fibril complex, and (ii) detection of a signal from the detectable label indicates the presence of one or more fibrils in the sample.

[0051] In some embodiments, the method comprises: (a) contacting a solid support comprising a fibril-specific antibody with a sample such that, if present in the sample, the fibrils bind to the antibody and are immobilized on the surface to form a complex; (b) detecting the complex.

[0052] Any solid support known in the art, including but not limited to those made of a polymer material in the form of a planar substrate or beads, can be used in the methods described herein. For example, the solid support can be a slide, a multiwell plate (e.g., a 96-well plate), or beads, such as latex, agarose, sepharose, streptavidin, tosyl-activated, epoxy, polystyrene, amino beads, amine beads, carboxyl beads, etc. In certain embodiments, the beads can be particles, e.g., microparticles. The terms "beads" and "particles" are used interchangeably herein and refer to substantially spherical solid supports. The terms "microparticles" and "microbeads" are used interchangeably herein and refer to microbeads or microparticles that are permitted to occupy or adhere to an array of wells, such as an array of wells in a detection module. A variety of techniques known in the art can be used to attach a protein or peptide to a solid support such as a plate or microparticle. For example, a wide variety of techniques for adding a reactive moiety to a protein are known, such as the method described in U.S. Patent No. 5,620,850. Methods for attachment of proteins to surfaces are also described, for example, in Heller, Acc. Chem. Res., 23:128 (1990).

[0053] The solid support can be contacted with a large amount of sample using any suitable method known in the art. As used herein, the term "contact" refers to any type of mixing action that sufficiently brings the solid support into proximity with one or more fibrils in the sample such that a binding interaction occurs when one or more fibrils are present in the sample. Contact can be achieved in a variety of different ways, including mixing the sample with a multi-well plate or microparticles. Contact can be repeated as many times as necessary. Incubation may be in a binding buffer that promotes specific binding interactions, such as albumin (e.g., BSA), non-ionic detergents (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). Other conditions for the binding interaction, such as temperature and salt concentration, may also be determined empirically or based on the manufacturer's instructions. For example, contact can be performed at room temperature (21°C to 28°C, e.g., 23°C to 25°C), 37°C, or 4°C. As used herein, the terms "detectable label" and "label" refer to a moiety that can generate a signal detectable by visual or instrumental means. The detectable label can be, for example, a signal-generating substance such as a chromogen, a fluorescent compound, an enzyme, a chemiluminescent compound, a radioactive compound, etc. In one embodiment, the detectable label can be a fluorescent compound such as a fluorophore. The presence or amount of fibrils in the sample can be determined (e.g., quantified) using any suitable method known in the art. Such methods include, but are not limited to, immunoassays such as ELISA.

[0054] In some embodiments, the invention relates to an assay for detecting liraglutide fibrils in excess of soluble liraglutide and / or monomeric liraglutide, comprising an antibody according to the invention, which antibody can detect liraglutide fibrils at a fibril concentration in solution of 1 to 1000 ppm, such as 1 to 10 ppm of fibrils, or 10 to 100 ppm of fibrils, or 100 to 1000 ppm of fibrils.

[0055] In some embodiments, "a" means "one or more". As used herein, the term "about" means a range of minus 10% to plus 10% of the referenced value. Unless otherwise indicated herein, terms presented in the singular also include plural situations.

[0056] Modes for Carrying Out the Invention Non-limiting embodiments of the present invention include the following. 1. An antibody that binds to liraglutide fibrils. 2. An antibody that binds to liraglutide fibrils, wherein the fibrils are prepared according to Assay (I) herein. 3. An antibody that binds to semaglutide fibrils. 4. An antibody that binds to semaglutide fibrils, wherein the fibrils are prepared according to Assay (II) herein. 5. The antibody according to embodiment 1 or 2, wherein the antibody has a detection limit for liraglutide fibrils at a concentration at least 10 times lower, such as at least 100 times or at least 1000 times lower, than the detection limit of liraglutide fibrils in a ThT assay, and the detection limit is optionally determined according to Assay (VI) herein. 6. The antibody according to embodiment 1 or 2, wherein the antibody has a binding level to liraglutide fibrils at least 10 times higher, such as at least 20 times higher or at least 50 times higher, than the binding level of the antibody to soluble liraglutide. 7. The antibody according to embodiment 1 or 2, wherein the antibody has a detection limit for liraglutide fibrils at a concentration at least 10 times lower, such as at least 100 times or at least 1000 times lower, than the detection limit of liraglutide fibrils in a ThT assay, and the detection limit is optionally determined according to Assay (VI-B) herein. 8. An antibody according to embodiment 1 or 2, wherein the antibody has a binding level to liraglutide fibrils that is at least 5-fold higher, such as 10-fold higher, than the binding level of the antibody to soluble liraglutide, and the binding level is optionally determined according to assay (III-B) herein. 9. An antibody according to embodiment 3 or 4, wherein the antibody has a detection limit for semaglutide fibrils at a concentration that is at least 10-fold lower, such as at least 100-fold lower or at least 1000-fold lower, than the detection limit of semaglutide fibrils in the ThT assay, and the detection limit is optionally determined according to assay (VI) herein. 10. An antibody according to embodiment 3 or 4, wherein the antibody has a binding level to semaglutide fibrils that is at least 10-fold higher, such as at least 20-fold higher or at least 50-fold higher, than the binding level of the antibody to soluble semaglutide. 11. An antibody according to embodiment 6 or 10, wherein the binding level is determined according to assay (IV) herein. 12. An antibody according to embodiment 6 or 10, wherein the binding level is determined according to assay (III) herein. 13. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. 14. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody a. SEQ ID NO: 37, 38, and 39, b. SEQ ID NO: 43, 44, and 45, c. SEQ ID NO: 49, 50, and 51, d. SEQ ID NO: 55, 56, and 57, e. SEQ ID NO: 61, 62, and 63, f. SEQ ID NO: 67, 68, and 69, g. SEQ ID NO: 73, 74, and 75, h. SEQ ID NOs: 79, 80, and 81, i. SEQ ID NOs: 85, 86, and 87, j. SEQ ID NOs: 91, 92, and 93, k. SEQ ID NOs: 97, 98, and 99, l. SEQ ID NOs: 103, 104, and 105, or an antibody comprising a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. 15. An antibody that binds to liraglutide fibril, wherein the variable region of the light chain of said antibody is a. SEQ ID NOs: 40, 41, and 42, b. SEQ ID NOs: 46, 47, and 48, c. SEQ ID NOs: 52, 53, and 54, d. SEQ ID NOs: 58, 59, and 60, e. SEQ ID NOs: 64, 65, and 66, f. SEQ ID NOs: 70, 71, and 72, g. SEQ ID NOs: 76, 77, and 78, h. SEQ ID NOs: 82, 83, and 84, i. SEQ ID NOs: 88, 89, and 90, j. SEQ ID NOs: 94, 95, and 96, k. SEQ ID NOs: 100, 101, and 102, l. SEQ ID NOs: 106, 107, and 108, or an antibody comprising a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. 16. An antibody that binds to liraglutide fibril, comprising the variable region of the heavy chain as described in any one of the preceding embodiments and the variable region of the light chain as described in any one of the preceding embodiments. 17. An antibody that binds to liraglutide fibrils, comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 18. An antibody that binds to liraglutide fibrils, having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. 19. An antibody that binds to liraglutide fibrils, comprising a variable light chain (VL) sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 20. An antibody that binds to liraglutide fibrils, comprising a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 21. The antibody according to any one of the preceding embodiments, wherein the antibody is an isolated antibody. 22. The antibody according to any one of the preceding embodiments, wherein the antibody is an isolated antibody. 23. The antibody according to any one of the preceding embodiments, wherein the antibody comprises an Fc domain. 24. The antibody according to any one of the preceding embodiments, wherein the antibody is a single-chain Fv fragment further comprising an Fc domain. 25. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. 26. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils. 27. An antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the semaglutide fibril. 28. Use of an antibody according to any one of the preceding embodiments for the identification of liraglutide fibrils or semaglutide fibrils. 29. Use of an antibody according to any one of the preceding embodiments as an affinity ligand for removing fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide, or (ii) semaglutide fibrils and soluble semaglutide. 30. A method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising: a) binding an antibody according to any one of the preceding embodiments to the liraglutide fibrils or semaglutide fibrils. 31. A method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising: a) binding an antibody according to any one of the preceding embodiments to the liraglutide fibrils or semaglutide fibrils. 32. The method according to any one of the preceding embodiments, further comprising: b) detecting the antibody bound to the liraglutide fibrils or semaglutide fibrils. 33. The method according to any one of the preceding embodiments, further comprising: c) optionally quantifying the antibody bound to the liraglutide fibrils or semaglutide fibrils by use of a standard of the fibrils. 34. The method according to any one of the preceding embodiments, wherein the fibrils are in solution. 35. The method according to any one of the preceding embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide. 36. The method according to any one of the preceding embodiments, wherein the fibrils are in a solution that further does not contain other peptides or proteins other than liraglutide fibrils and optionally soluble liraglutide. 37. An antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to assay (I) herein, and the antibody has a. a binding level to liraglutide fibrils that is at least 10-fold higher, such as at least 20-fold higher or at least 50-fold higher, than the binding level of the antibody to soluble liraglutide, and / or b. a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower, than the detection limit of liraglutide fibrils in the ThT assay, and the detection limit is optionally determined according to assay (VI) herein. 38. An antibody that binds to semaglutide fibrils, wherein the fibrils are optionally prepared according to assay (II) herein, and the antibody has c. a binding level to semaglutide fibrils that is at least 10-fold higher, such as at least 20-fold higher or at least 50-fold higher, than the binding level of the antibody to soluble semaglutide, and / or d. a detection limit for semaglutide fibrils at a concentration that is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower, than the detection limit of semaglutide fibrils in the ThT assay, and the detection limit is optionally determined according to assay (VI) herein. 39. The antibody according to embodiment 37 or 38, wherein the binding level is determined according to assay (IV) herein. 40. The antibody according to embodiment 37 or 38, wherein the binding level is determined according to assay (III) herein. 41. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NOs: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 115, and 121, or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. 42. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. 43. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody is a. SEQ ID NO: 37, 38, and 39, b. SEQ ID NO: 43, 44, and 45, c. SEQ ID NO: 49, 50, and 51, d. SEQ ID NO: 55, 56, and 57, e. SEQ ID NO: 61, 62, and 63, f. SEQ ID NO: 67, 68, and 69, g. SEQ ID NO: 73, 74, and 75, h. SEQ ID NO: 79, 80, and 81, i. SEQ ID NO: 85, 86, and 87, j. SEQ ID NO: 91, 92, and 93, k. SEQ ID NO: 97, 98, and 99, l. SEQ ID NO: 103, 104, and 105, or an antibody comprising CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of any of the sequences having one, two, or three amino acid substitutions, deletions, or insertions. 44. An antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody is a. SEQ ID NO: 40, 41, and 42, b. SEQ ID NO: 46, 47, and 48, c. SEQ ID NO: 52, 53, and 54, d. SEQ ID NO: 58, 59, and 60, e. SEQ ID NO: 64, 65, and 66, f. SEQ ID NO: 70, 71, and 72, g. SEQ ID NO: 76, 77, and 78, h. SEQ ID NO: 82, 83, and 84, i. SEQ ID NOs: 88, 89, and 90, j. SEQ ID NOs: 94, 95, and 96, k. SEQ ID NOs: 100, 101, and 102, l. SEQ ID NOs: 106, 107, and 108, or a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. An antibody comprising the same. 45. An antibody that binds to liraglutide fibril, comprising the variable region of the heavy chain described in any one of the preceding embodiments and the variable region of the light chain described in any one of the preceding embodiments. An antibody. 46. An antibody that binds to liraglutide fibril, comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. An antibody. 47. An antibody that binds to liraglutide fibril, having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. An antibody. 48. The antibody according to any one of the preceding embodiments, wherein the antibody is an isolated antibody. 49. The antibody according to any one of the preceding embodiments, wherein the antibody is a single-chain Fv fragment. 50. The antibody according to any one of the preceding embodiments, wherein the antibody comprises an Fc domain. 51. The antibody according to any one of the preceding embodiments, wherein the antibody is a single-chain Fv fragment further comprising an Fc domain. 52. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibril and / or the semaglutide fibril. 53. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibril. 54. An antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the semaglutide fibril. 55. Use of an antibody according to any one of the preceding embodiments for the identification of liraglutide fibrils or semaglutide fibrils. 56. Use of an antibody according to any one of the preceding embodiments as an affinity ligand for removing fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide, or (ii) semaglutide fibrils and soluble semaglutide. 57. A method for identifying liraglutide fibrils or semaglutide fibrils, comprising: a) binding an antibody according to any one of the preceding embodiments to liraglutide fibrils or semaglutide fibrils. 58. A method for quantifying liraglutide fibrils or semaglutide fibrils, comprising: a) binding an antibody according to any one of the preceding embodiments to liraglutide fibrils or semaglutide fibrils. 59. The method according to any one of the preceding embodiments, further comprising: b) detecting the antibody bound to liraglutide fibrils or semaglutide fibrils. 60. The method according to any one of the preceding embodiments, further comprising: c) optionally quantifying the antibody bound to liraglutide fibrils or semaglutide fibrils by use of a standard of the fibrils. 61. The method according to any one of the preceding embodiments, wherein the fibrils are in solution. 62. The method according to any one of the preceding embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide. 63. The method according to any one of the preceding embodiments, wherein the fibrils are in a solution that further does not contain other peptides or proteins other than liraglutide fibrils and optionally soluble liraglutide. 64. An antibody that binds to liraglutide fibrils, wherein the variable region of the heavy chain of the antibody is m. Sequence numbers 115, 116, and 117, n. Sequence numbers 121, 122, 123, or an antibody comprising a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. 65. An antibody that binds to liraglutide fibril, wherein the variable region of the light chain of the antibody is aa. Sequence numbers 118, 119, and 120, bb. Sequence numbers 124, 125, and 126, or an antibody comprising a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of any of said sequences having one, two, or three amino acid substitutions, deletions, or insertions. 66. An antibody that binds to liraglutide fibril, comprising the variable region of the heavy chain described in any one of the preceding embodiments and the variable region of the light chain described in any one of the preceding embodiments. 67. An antibody that binds to liraglutide fibril, comprising a sequence selected from the group consisting of sequence numbers 109 and 110, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 68. An antibody that binds to liraglutide fibril, having at least 80% sequence identity, such as at least 90% or at least 95%, to a sequence selected from the group consisting of sequence numbers 109 and 110. 69. An antibody according to any one of embodiments 37 to 68, that binds to liraglutide fibril and comprises a variable light chain (VL) sequence selected from the group consisting of sequence numbers 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 70. An antibody that binds to liraglutide fibrils and comprises a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114, or any of said sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10, the antibody according to any one of embodiments 37 to 69. 71. The antibody according to any one of embodiments 37 to 70, wherein the antibody is an isolated antibody. 72. The antibody according to any one of embodiments 37 to 71, wherein the antibody is a single-chain Fv fragment. 73. The antibody according to any one of embodiments 37 to 72, wherein the antibody comprises an Fc domain. 74. The antibody according to any one of embodiments 37 to 73, wherein the antibody is a single-chain Fv fragment further comprising an Fc domain. 75. The antibody according to any one of embodiments 37 to 74, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. 76. The antibody according to any one of embodiments 37 to 75, wherein the antibody specifically binds to the liraglutide fibrils. 77. The antibody according to any one of embodiments 37 to 76, wherein the antibody specifically binds to the semaglutide fibrils. 78. The fibrils are optionally prepared according to assay (I) herein, and the antibody a. a binding level to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, determined according to assay (III) herein at a liraglutide fibril concentration of at least 25 μM, and / or b. a detection limit for liraglutide fibrils at a concentration that is at least 10-fold lower than the detection limit of liraglutide fibrils in a ThT assay, determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM, and / or c. A binding level to liraglutide fibrils that is at least 5-fold higher than the binding level of the antibody to soluble liraglutide, wherein the antibody has a monomer purity of greater than 95%, and the binding level is determined according to the assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM, and / or d. A detection limit of liraglutide fibrils at a concentration at least 10-fold lower than the detection limit of liraglutide fibrils in the ThT assay, wherein the antibody has a monomer purity of greater than 95%, and the detection limit is determined according to the assay (VI-B) herein at a liraglutide fibril concentration of at least 0.025 μM, an antibody according to any one of embodiments 37 to 77 having the detection limit. 79. An antibody according to any one of embodiments 37 to 78, wherein the antibody is capable of detecting liraglutide fibrils at a fibril concentration of 1 to 1000 ppm in solution, such as 1 to 10 ppm of fibrils, or 10 to 100 ppm of fibrils, or 100 to 1000 ppm of fibrils. 80. An antibody according to any one of embodiments 37 to 79, wherein the antibody has a monomer purity of greater than 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%. 81. An antibody according to any one of embodiments 37 to 80, wherein the antibody has a monomer purity of greater than 95%. 82. Use of an antibody according to any one of embodiments 37 to 81 for the identification of liraglutide fibrils or semaglutide fibrils. 83. Use of an antibody according to any one of embodiments 37 to 81 as an affinity ligand for removing fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide, or (ii) semaglutide fibrils and soluble semaglutide. 00 84. A method for identifying liraglutide fibrils or semaglutide fibrils, comprising a) binding an antibody according to any one of embodiments 37 to 81 to liraglutide fibrils or semaglutide fibrils. 85. A method for quantifying liraglutide fibrils or semaglutide fibrils, comprising: a) binding an antibody according to any one of Embodiments 37 to 81 to liraglutide fibrils or semaglutide fibrils. 86. 86. The method according to Embodiment 84 or 85, further comprising: b) detecting the antibody bound to the liraglutide fibrils or semaglutide fibrils. 87. 87. The method according to any one of Embodiments 84 to 86, further comprising: c) optionally quantifying the antibody bound to the liraglutide fibrils or semaglutide fibrils by using a standard of the fibrils. 88. The method according to any one of Embodiments 84 to 87, wherein the fibrils are in solution. 89. The method according to any one of Embodiments 84 to 88, wherein the fibrils are in a solution further comprising soluble liraglutide. 90. The method according to any one of Embodiments 84 to 89, wherein the fibrils are in a solution that does not further contain other peptides or proteins other than liraglutide fibrils and optionally soluble liraglutide. 91. An assay for selectively detecting liraglutide fibrils over soluble liraglutide and / or monomeric liraglutide, comprising an antibody according to any one of Embodiments 37 to 81, wherein the antibody can detect liraglutide fibrils at a fibril concentration of 1 to 1000 ppm in solution, such as 1 to 10 ppm of fibrils, or 10 to 100 ppm of fibrils, or 100 to 1000 ppm of fibrils.

Examples

[0057] List of Abbreviations · PBS: Phosphate Buffered Saline (aqueous solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 adjusted to pH 7.4) · PES: Polyethersulfone · scFv-Fc: Single-chain variable fragment linked to the Fc domain · ThT: Thioflavin T

[0058] Materials and Methods Preparation, Screening, and Cloning of an Antibody Library of Selected Antibody Variants Antibodies were isolated through two steps of library screening. In the first step of screening, single-chain variable fragments (scFvs) that generated a single-chain variable fragment (scFv) yeast surface display library by the diversity of the heavy-chain complementarity-determining region 3 (HCDR3) of 4D5 scFv were genetically fused to the C-terminus of the yeast Aga2 protein via a flexible linker to enable antibody display on the cell surface. The yeast display antibody library was screened by binding to liraglutide fibrils (and soluble liraglutide as a control) immobilized on magnetic beads (Dynabeads M-280 Tosylactivated, 14203, Invitrogen). To prepare the beads, 8×10 7 individual beads were first washed (2×) with 1 mL of sterile PBS. Soluble liraglutide (100 μg, from a 6 mg / mL stock in the drug composition buffer) was diluted in PBS containing the magnetic beads (final volume 800 μL) and bound to the beads overnight (4 °C without stirring). For the beads coated with fibrillar liraglutide, 100 μg of liraglutide fibrils were bound to the beads in 800 μL of PBS at room temperature overnight with rotary mixing. The next day, the beads were washed (2×) with 1 mL of PBS supplemented with 10 mM glycine to quench the unreacted tosyl groups on the beads, and then washed (2×) with 1 mL of PBS supplemented with 1 g / L BSA (PBS-B) and then incubated with yeast. Eight positive selections were performed against the beads coated with liraglutide fibrils in PBS-B supplemented with 1% milk. To isolate yeast with conformation-specific antibodies against liraglutide fibrils, the last three selections incorporated negative selections performed against the beads coated with soluble liraglutide in PBS-B prior to the positive selection against liraglutide fibrils.

[0059] In the second stage of library screening (affinity maturation), a sub-library was designed for one of the best clones from the first stage of screening. The second-generation library diversified the sites in LCDR1, LCDR3, and HCDR2. This library was subjected to four rounds of selection against relugolix fibrils. The first two rounds of screening incorporated two consecutive negative selections against soluble relugolix (immobilized on magnetic beads) prior to positive selection against immobilized relugolix fibrils. The negative selections were performed in PBS-B, while the positive selections were carried out in PBS-B supplemented with 1% milk. Also, three consecutive negative selections were performed in the third and fourth rounds against beads coated with glucagon fibrils. Beads coated with glucagon fibrils were prepared as previously described (Stimple et al., 2019).

[0060] The selected antibodies were cloned into the mammalian expression vector anti-Notch1_E6-pBIOCAM5 as previously described (Stimple et al., 2019). Briefly, the insert and backbone plasmid were digested with NcoI and NotI, purified, and ligated. The insertion of the scFv coding fragment was confirmed by Sanger sequencing. These plasmids express a bivalent scFv-human Fc fusion protein with a 6xHis tag and a 3xFLAG tag on the C-terminus of the antibody.

[0061] Antibody expression and purification Proteins were expressed in the Expi293F expression system (catalog number A14635). Expi293F cells were subcultured and grown until the cell density reached approximately 3 - 5 million viable cells per milliliter. Plasmid (30 μg) was transfected into 25 mL of Expi293 cells. The complex of ExpiFectamine 293 and plasmid DNA was prepared as described in the manufacturer's guidance. Briefly, plasmid DNA and ExpiFectamine reagent were diluted in Opti-MEM medium and mixed via gentle pipetting. After a 5-minute incubation, the diluted transfection reagent was mixed with the diluted DNA. The complex of transfection reagent and DNA was incubated at room temperature for 20 minutes and added to Expi293 cells. The cells were incubated at 37 °C and 5% CO2 with shaking. According to the manufacturer's instructions, enhancer 1 and 2 solutions were added to the cells 20 hours after transfection. Three days later, the medium containing the secreted antibody was harvested and centrifuged at 3400×g for 45 minutes to remove cells and related debris.

[0062] Antibodies were purified using Protein A chromatography. Protein A beads (20334, Thermo Fisher Scientific) were washed with PBS and incubated with glycine buffer (pH 2.5) for 20 minutes. Next, the beads were washed with PBS, then 0.5 mL of the beads were added to 30 mL of purification medium and incubated overnight at 4 °C. The next day, the medium with Protein A beads was added to a 10 mL purification column (89898, Thermo Fisher Scientific). The beads were collected via vacuum filtration and washed thoroughly with PBS (100 mL). Then, the Protein A beads were incubated with 2 mL of 0.1 M glycine buffer (pH 3.0) for 15 minutes, and the buffer (containing the eluted protein) was collected by centrifugation. Next, the eluted antibody was buffer-exchanged into PBS using Zeba Spin Desalting Columns (89891, Thermo Fisher Scientific). Protein concentration was assayed via absorbance measurement at 280 nm (extinction coefficient of 168,460~205,360 M -1 cm -1 .

[0063] Size exclusion chromatography Analytical and preparative size exclusion chromatography (SEC) experiments were performed using a Shimadzu Prominence HPLC System. The running buffer was 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 200 mM arginine. The column flow rate was 0.75 mL / min. An antibody sample (0.1 mg / mL) was injected (100 μL) into the column (GE 28990944, Superdex 200 Increase 10 / 300 GL column, inner diameter 10 mm, length 300 mm), and absorbance signals were monitored at 220 and 280 nm. For preparative SEC, monomer fractions were isolated using an FRC-10A fraction collector.

[0064] Assay (I): Preparation of liraglutide fibrils A test solution of liraglutide at 6 mg / mL was prepared in drug composition buffer (14.0 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium hydrogen phosphate dihydrate), and the final pH was adjusted to 8.15 using NaOH and / or HCl as necessary, followed by syringe filtration (0.22 μm PES filter). An aliquot of 1 mL of the liraglutide solution was dispensed into microcentrifuge tubes, a single 3 mm glass bead (Sigma Z265926) was added to each tube, and the tubes were incubated in a thermomixer at 37 °C with orbital shaking at 300 rpm for 15 - 20 days.

[0065] Fibril formation was monitored by removing a small sample (about 75 μL) of the test solution from the tubes using a positive ThT signal, and this was analyzed according to assay (V) (ThT assay) described herein. If the sample showed fluorescence at least 5 - fold higher than that of a freshly prepared test solution in assay (V) (ThT assay) herein, the fibrils were precipitated at 221,000×g (1 hour, 4 °C). Fibrils, e.g., gel - like fibrils, were observed at the bottom of the tubes. The supernatant was removed from the tubes (the supernatant was retained for analysis by assay (VII) (BCA assay) herein). The pellet was gently washed once with drug composition buffer at pH 8.15 (without disturbing the pellet), then resuspended in the original volume of drug composition buffer at pH 8.15 (taking into account any volume removed for ThT analysis), and stored at 4 °C. The concentration of the fibrils was determined according to assay (VII) herein. For this calculation to be accurate, it is important to resuspend the fibril pellet in exactly the same total volume after centrifugation.

[0066] Assay (II): Preparation of semaglutide fibrils A test solution of 6 mg / mL semaglutide adjusted to pH 6.9 (using NaOH and / or HCl as needed) and optionally containing 50 mM NaCl, and a drug composition buffer (14.0 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium hydrogen phosphate dihydrate) was placed in a microcentrifuge tube in 1 mL aliquots, a single 3 mm glass bead (Sigma Z265926) was added to each tube, and the tubes were incubated in a 37 °C thermomixer for 15 - 20 days with orbital shaking at 300 rpm.

[0067] Fibril formation was monitored by removing a small sample (about 75 μL) of the test solution from the tube using a positive ThT signal and analyzing this according to assay (V) (ThT assay) described herein. If the sample showed fluorescence at least 5 - fold higher than freshly prepared semaglutide in the drug composition buffer at pH 6.9 in assay (V) (ThT assay), the fibrils were precipitated at 221,000×g (1 hour, 4 °C). Fibrils, e.g., gel - like fibrils, were observed at the bottom of the tube. The supernatant was removed from the tube (the supernatant was retained for analysis by assay (VII) (BCA assay)). The pellet was gently washed once with the drug composition buffer at pH 6.9 (without disturbing the pellet), then resuspended in the original volume of the drug composition buffer at pH 6.9 (taking into account any volume removed for ThT analysis) and stored at 4 °C. The concentration of the fibrils was determined according to assay (VII) herein. For this calculation to be accurate, it is important to resuspend the fibril pellet in exactly the same total volume after centrifugation.

[0068] Assay (III): Antibody Specificity Ratio (Method 1) The antibody specificity ratio was determined as follows: 1. ELISA Plate Preparation (3 plates): a. For fibril-coated ELISA plates: The liraglutide fibrils prepared according to Assay (I) herein were resuspended in the drug composition buffer (having a fibril concentration determined by Assay (VII) (BCA assay) herein), and about 300 μL of the sample was sonicated on ice in a microcentrifuge tube (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) to obtain. The solution was diluted to 25 μM liraglutide fibrils in PBS, and 100 μL of the sample was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). b. For soluble liraglutide-coated plates: 6 mg / mL liraglutide was solubilized in the drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) adjusted to pH 8.15 using NaOH and / or HCl as needed, and the solution was filtered through a 0.22 μm PES filter. The solution was diluted to 25 μM liraglutide in PBS, and 100 μL of the sample was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). i. For the "background" plates: 100 μL of PBS was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). 2. Cover the plate with an adhesive film, wrap it with aluminum foil, and incubate overnight at 4°C. 3. The next day, the plate was washed three times by adding 300 μL of PBS to each well. 4. The plate was blocked by adding 300 μL of PBS supplemented with 0.1% Tween 20 and 10 g / L BSA to each well. Then, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 3 hours. 5. While the plates were blocked, the antibody samples were spun in a centrifuge at 21,000×g for 5 minutes, and the concentration of the supernatant was measured by absorbance at 280 nm. Each antibody was serially diluted to 5 nM in PBS supplemented with 0.1% Tween20 and 1 g / L BSA. 6. The plates were washed three times by adding 300 μL of PBS to each well. 7. 100 μL of the antibody solution was dispensed into each well. Each antibody was tested in duplicate (i.e., 2 wells per antibody per plate). The plates were then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. 8. A secondary antibody solution was prepared by diluting the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 in PBS supplemented with 0.1% Tween20 and 10 g / L BSA. 9. The plates were washed three times by adding 300 μL of PBS to each well. 10. 100 μL of the secondary antibody solution was dispensed into each well. The plates were then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34208) was taken out of the refrigerator, the solution was equilibrated to room temperature, and 2 M (4 N) H2SO4 was prepared. 11. The plates were washed three times by adding 300 μL of PBS to each well. 12. 100 μL of Ultra-TMB was added to each well and incubated until a yellow product was formed (5 - 10 minutes). 13. The reaction was quenched by adding 100 μL of 2 M H2SO4. 14. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). Calculation: The ratio of the ELISA signal (absorbance at 450 nm) was calculated for each antibody against the fibril-coated plate and its signal against the soluble liraglutide-coated plate and the background plate. These ratios are the fibril / soluble ratio and fibril / background ratio reported herein. For example, if the antibody gave signals of 1.5 for liraglutide fibril, 0.05 for soluble liraglutide, and 0.1 for the background plate, the fibril / soluble ratio is 1.5 / 0.05 = 30, and the fibril / background ratio is 1.5 / 0.1 = 15.

[0069] Assay (III-B): Antibody Specificity Ratio (Method 1B) 1. The night before the assay: BSA was solubilized at 1 mg / mL in PBS and then sterilized through a 0.22 μm PES filter using a 30 cc Luer-lock syringe, and 150 μL of the solution was dispensed into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. The plate was covered with an adhesive film, wrapped with aluminum foil, and incubated overnight at 4°C. 2. The ELISA plate (coated with BSA) was removed from the refrigerator and the wells were washed three times with 300 μL of PBS. a. For the fibril-coated ELISA plate: The liraglutide fibrils prepared according to Assay (I) herein were resuspended in the drug composition buffer (having a fibril concentration determined by Assay (VII) (BCA assay) herein), and approximately 300 μL of the sample was sonicated on ice in a microcentrifuge tube (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) to obtain. The solution was diluted to 10 μM liraglutide fibrils in PBS, and 100 μL of the sample was dispensed into each well. b. For the soluble liraglutide-coated plate: Liraglutide at 6 mg / mL was solubilized in a drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) adjusted to pH 8.15 using NaOH and / or HCl as needed, and the solution was filtered through a 0.22 μm PES filter. The solution was diluted to 10 μM liraglutide in PBS, and 100 μL of the sample was dispensed into each well. i. For the "background" plate: 100 μL of PBS was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). 3. The plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 3 hours without stirring. 4. During this 3-hour period, the antibody samples were spun in a centrifuge at 21,000 × g for 5 minutes, and the concentration of the supernatant was measured by absorbance at 280 nm. Each antibody was serially diluted to 5 nM in PBS supplemented with 0.1% Tween20 and 1 g / L BSA (unless otherwise specified for the concentration). 5. The plate was washed three times by adding 300 μL of PBS to each well. 6. 100 μL of the antibody solution was dispensed into each well. Then, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. 7. A secondary antibody solution was prepared by diluting a secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 in PBS supplemented with 0.1% Tween20 and 10 g / L BSA. 8. The plate was washed three times by adding 300 μL of PBST (PBS supplemented with 0.1% Tween20) to each well. 9. 100 μL of the secondary antibody solution was dispensed into each well. Then, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) was taken out of the refrigerator, the solution was equilibrated to room temperature, and 2 M (4 N) H2SO4 was prepared. 10. The plate was washed three times by adding 300 μL of PBST to each well. 11. 100 μL of Ultra-TMB was added to each well and incubated until a yellow product was formed (5 - 10 minutes). 12. The reaction was quenched by the addition of 100 μL of 2 M H2SO4. 13. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). Calculation: The ratio of the ELISA signal (absorbance at 450 nm) was calculated for each antibody against the fibril-coated plate and its signal against the soluble liraglutide-coated plate and the background plate. These ratios are the fibril / soluble ratio and fibril / background ratio reported herein. For example, if an antibody gave a signal of 1.5 for liraglutide fibrils, 0.05 for soluble liraglutide, and 0.1 for the background plate, the fibril / soluble ratio would be 1.5 / 0.05 = 30, and the fibril / background ratio would be 1.5 / 0.1 = 15.

[0070] Assay (IV): Antibody Specificity Ratio (Method 2) The antibody specificity ratio was determined as follows: Before the assay: Solubilize BSA at 1 mg / mL in PBS, then sterilize it through a 0.22 μm PES filter using a 30 cc Luer-lock syringe, and dispense 150 μL of the solution into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. Cover the plate with an adhesive film, wrap it with aluminum foil, and incubate overnight at 4 °C.

[0071] On the day of the assay: 1. Solubilize liraglutide at 60 mg / mL in a drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) adjusted to pH 8.15 using NaOH and / or HCl as necessary, and filter it through a 0.22 μm PES filter. Dilute the solution to 6 mg / mL (1600 μM) liraglutide in PBS. This solution is referred to as the "soluble liraglutide solution". 2. Liraglutide fibrils (hereinafter referred to as fibrils), obtained according to Assay (I) herein and resuspended in a drug composition buffer at pH 8.15 in a sample of approximately 300 μL (having a fibril concentration determined by Assay (VII) (BCA assay) herein), were sonicated on ice in a microcentrifuge tube (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) to obtain a solution. This solution is referred to as the "fibril solution". 3. Dilute the sonicated fibril solution into the soluble liraglutide solution such that the final concentration of fibrils is 100 μM. Then, further serially dilute the solution in the soluble liraglutide solution to samples resulting in 0.1 μM fibrils. Two controls were used: i) PBS (peptide-free), and ii) fibril-free (soluble liraglutide solution only). 4. Remove the ELISA plate (coated with BSA) from the refrigerator and wash the wells three times with 300 μL of PBS. 5. 100 μL of each sample or control from (3) above was dispensed into the wells of a freshly washed plate, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 3 hours without stirring. 6. During the 3-hour incubation above, approximately 75 μL of the antibody to be tested (e.g., scFv-Fc fusion protein) was spun at 21,000×g for 5 minutes to precipitate any particulate matter. The supernatant was removed, and the A280nm of this supernatant was determined to calculate the antibody concentration. The antibody was serially diluted to 5 nM in PBS + 0.1% Tween20 + 1 g / L BSA and kept on ice until use. 7. At the end of the 3-hour incubation, the wells of the plate were washed three times with 300 μL of PBS. 8. 100 μL of 5 nM antibody was added to each well. The plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. 9. During the 1-hour incubation above, the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) was diluted 1:1000 in PBS + 0.1% Tween20 + 10 g / L BSA. 10. At the end of the 1-hour incubation, the wells were washed three times with 300 μL of PBS. 11. 100 μL of the secondary antibody solution was added to each well. The plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. 12. During the secondary antibody incubation, 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) was taken out of the refrigerator and the solution was equilibrated to room temperature. 2 M (4 N) H2SO4 was prepared. 13. At the end of the 1-hour incubation, the wells were washed three times with 300 μL of PBS. 14. 100 μL of Ultra-TMB was added to each well and incubated for 10 minutes. 15. The reaction was quenched by the addition of 100 μL of 2 M H2SO4. 16. The absorbance at 450 nm was read in a microplate reader (BioTek Synergy Neo). Calculation: The ELISA signal for each antibody in the wells containing fibrils was divided by the ELISA signal for the same antibody in control wells having soluble liraglutide and no fibrils. This ratio is the mixture / soluble specificity ratio. For example, if a given antibody gave a signal of 1.5 for 0.1 μM liraglutide fibrils in a mixture with soluble liraglutide and a signal of 0.05 for soluble liraglutide (lacking fibrils), the mixture / soluble specificity ratio is 1.5 / 0.05 = 30.

[0072] Assay (V): ThT assay The test solution of the peptide was analyzed for the presence of fibrils immediately after fibril formation. The peptide concentration before fibril formation was 6 mg / mL. The peptide may be liraglutide or semaglutide. Liraglutide fibrils can be prepared according to Assay (I) herein. Semaglutide fibrils can be prepared according to Assay (II) herein. A 75 μL sample of the test solution was mixed with 1.36 μL of the ThT stock solution (stock concentration: 2200 μM ThT) to reach a final ThT concentration of 40 μM in the peptide / ThT mixture. For liraglutide, the final concentration in this mixture was 1571 μM liraglutide (calculated before fibrillization). A 50 μL sample of the peptide / ThT mixture was added to the wells of a black 384-well plate (Fisherbrand 384 Well Polystyrene Plates, 12566624, Thermo Fisher Scientific), and after 5 - 10 minutes, the ThT fluorescence (λex = 444 nm, λem = 482 nm) values were measured using a Biotek Synergy Neo microplate reader.

[0073] Assay (VI): ThT assay compared with the antibody assay The detection of liraglutide fibrils in a mixture with soluble liraglutide was determined using a ThT detection method compared to an antibody assay. 1. Liraglutide was solubilized at 60 mg / mL in a drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) adjusted to pH 8.15 using NaOH and / or HCl as needed, and filtered through a 0.22 μm PES filter. The solution was diluted to 6 mg / mL (1600 μM) liraglutide in PBS. This solution is referred to as the "soluble liraglutide solution". 2. Liraglutide fibrils (hereinafter fibrils) obtained according to Assay (I) herein and resuspended in a drug composition buffer at pH 8.15 in a sample of approximately 300 μL (having a fibril concentration determined by Assay (VII) (BCA assay) herein) were sonicated on ice in a microcentrifuge tube (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) to obtain a solution referred to as the "fibril solution". 3. The sonicated fibril solution was diluted into the soluble liraglutide solution such that the final concentration of fibrils was 100 μM. The solution was then further serially diluted into the soluble liraglutide solution to samples resulting in fibril concentrations of 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM. Two controls were used: i) PBS (peptide-free), and ii) fibril-free (soluble liraglutide solution only). a. At this point, the solution obtained by diluting the fibril solution into the soluble liraglutide solution (from Step 3) was added (100 μL) to the wells of an ovalbumin-coated ELISA plate and incubated at room temperature for 2 hours prior to ELISA detection using the antibody of the present invention. The remaining ELISA protocol was performed as described in Assay (IV) (from Step 7 onwards). 4. The residual samples (mixture of fibrils and soluble liraglutide, and PBS control and soluble liraglutide control from step 3) were incubated in microcentrifuge tubes for 2.5 hours at room temperature. 5. A stock solution of Thioflavin T (ThT) was prepared at a concentration of 2200 μM. ThT was added to the samples (initially at a total peptide concentration of 1600 μM) until a final concentration of 40 μM was reached, and 50 μL samples of the peptide / ThT mixture were added to the wells of a black 384-well plate (Fisherbrand 384 Well Polystyrene Plates, 12566624, Thermo Fisher Scientific). Using a Biotek Synergy Neo microplate reader, ThT fluorescence (λex = 444 nm, λem = 482 nm) values were measured for each sample. The final (total) peptide concentration in the peptide / ThT mixture was 1571 μM (calculated prior to fibrilization). 6. The fluorescence measurement values of the solution containing fibrils were divided by the fluorescence measurement values of the soluble liraglutide solution (control without fibrils), and the ratio was reported as the mixture / soluble ratio.

[0074] Assay (VI-B): ThT assay compared with antibody assay The detection of liraglutide fibrils in mixtures with soluble liraglutide was determined using a ThT detection method compared with an antibody assay. 1. The night before the assay: BSA was solubilized in PBS at 1 mg / mL, then sterilized through a 0.22 μm PES filter using a 30 cc Luer-lock syringe, and 150 μL of the solution was dispensed into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. The plate was covered with an adhesive film, wrapped in aluminum foil, and incubated overnight at 4 °C. 2. On the day of the assay, the liraglutide mixture was immobilized on the BSA-coated plate. a. Liraglutide was solubilized at 60 mg / mL in a drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) adjusted to pH 8.15 using NaOH and / or HCl as needed, and filtered through a 0.22 μm PES filter. The solution was diluted in PBS to 6 mg / mL (1600 μM) liraglutide. This solution is referred to as the "soluble liraglutide solution". b. Liraglutide fibrils (hereinafter referred to as fibrils), obtained according to assay (I) herein and resuspended in a drug composition buffer at pH 8.15 in a sample of approximately 300 μL (having a fibril concentration determined by assay (VII) (BCA assay) herein), were sonicated in a microcentrifuge tube on ice (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) to obtain a solution. This solution is referred to as the "fibril solution". c. The sonicated fibril solution was diluted into the soluble liraglutide solution such that the final concentration of fibrils was 100 μM. The solution was then further serially diluted in the soluble liraglutide solution to samples resulting in fibrils at 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM. Two controls were used: i) PBS (peptide-free), and ii) fibril-free (soluble liraglutide solution only). d. The solution obtained by diluting the fibril solution into the soluble liraglutide solution was added (100 μL) to the wells of a BSA-coated ELISA plate and incubated at room temperature for 3 hours. 3. The remaining ELISA protocol was performed with some modifications as described for assay (VI). a. During this 3-hour period, the antibody samples were spun in a centrifuge at 21,000 × g for 5 minutes and the concentration of the supernatant was measured by absorbance at 280 nm. Each antibody was serially diluted to 50 nM in PBS supplemented with 0.1% Tween20 (PBST). b. The plate was washed three times by adding 300 μL of PBS to each well. c. 100 μL of the antibody solution was dispensed into each well. Then, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. d. A secondary antibody solution was prepared by diluting a secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 in PBS supplemented with 0.1% Tween20 and 10 g / L of BSA. e. The plate was washed three times by adding 300 μL of PBST to each well. f. 100 μL of the secondary antibody solution was dispensed into each well. Then, the plate was covered with an adhesive film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34208) was taken out of the refrigerator, the solution was equilibrated to room temperature, and 2 M (4 N) H2SO4 was prepared. g. The plate was washed three times by adding 300 μL of PBST to each well. h. 100 μL of Ultra-TMB was added to each well and incubated until a yellow product was formed (5 - 10 minutes). i. The reaction was quenched by adding 100 μL of 2 M H2SO4. j. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). 4. Residual samples (a mixture of fibril and soluble liraglutide, as well as PBS control and soluble liraglutide control from step 3) were incubated in microcentrifuge tubes at room temperature for 2.5 hours. 5. A stock solution of thioflavin T (ThT) was prepared at a concentration of 2200 μM. ThT was added to the sample (initially at a total peptide concentration of 1600 μM) until a final concentration of 0.4 μM was reached, and 50 μL of the peptide / ThT mixture sample was added to the wells of a black 384-well plate (Fisherbrand 384 Well Polystyrene Plates, 12566624, Thermo Fisher Scientific). Using a Biotek Synergy Neo microplate reader, the ThT fluorescence (λex = 444 nm, λem = 482 nm) values were measured for each sample. The final (total) peptide concentration in the peptide / ThT mixture was 1571 μM (calculated before fibrillation). 6. The fluorescence measurement values of the solution containing fibrils were divided by the fluorescence measurement values of the soluble liraglutide solution (control without fibrils), and the ratio was reported as the mixture / soluble ratio.

[0075] Assay (VII): BCA Assay Using liraglutide as the standard for liraglutide fibrils and semaglutide as the standard for semaglutide fibrils (not BSA), the concentration of fibrils (e.g., liraglutide fibrils) was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Since phenol from the drug composition buffer reacts with the BCA reagent to varying degrees depending on the sample dilution, controls were run to account for the background signal arising from phenol for quantification. The concentration of fibrils in the resuspended fibril solution from Assay (I) or Assay (II) herein was determined by subtracting the peptide concentration in the supernatant (after ultracentrifugation) from the initial concentration (6 mg / mL) in the fibril aggregate. The analysis of liraglutide fibrils was performed as described below: 1. Liraglutide was dissolved in a drug composition buffer at 6 mg / mL (6000 μg / mL) and pH 8.15. This was designated as the "soluble liraglutide solution". 2. For the standards, the soluble liraglutide solution was diluted from (1) into PBS at concentrations of 2000, 1500, 1000, 750, 500, 250, 125, 25, and 0 μg / mL, and a “blank” containing the same volume of drug composition buffer diluted in PBS and lacking peptides but having pH 8.15 was prepared. For example, to prepare a 600 μg / L standard from 2000 μg / mL, 200 μL of the solution from (1) and 400 μL of PBS are required. To prepare the blank, 200 μL of the drug composition buffer (without peptide) and 400 μL of PBS were mixed. 3. The supernatant from the ultracentrifuged fibrils (in drug composition buffer, pH 8.15) obtained from assay (I) herein was diluted into PBS at the following dilutions: 1:2, 1:4, 1:8, 1:16, 1:32. For these samples containing drug composition buffer diluted in PBS at the same dilutions and having pH 8.15, “blanks” were also prepared. 4. To each well of a clear (non-binding or low-binding) flat-bottom 96-well plate, 10 μL of the standard (and a separate well with the corresponding blank), and 10 μL of the diluted sample (and a separate well with the corresponding blank) were added. 5. BCA Working reagent was made and 225 μL was added to each well, and the plate was covered with an adhesive film. 6. The plate was incubated at 37 °C until sufficient purple color formation occurred (this generally occurs relatively rapidly, and some color is often visible in some samples almost immediately). 7. The absorbance was read with a plate reader at 562 nm. 8. The “blank” absorbance values for the standards were subtracted from the values for the standards. A standard curve was fitted to the resulting (background-subtracted) absorbance by plotting the peptide concentration versus absorbance and fitting a second-order polynomial. 9. The "blank" absorbance value for the supernatant was subtracted from the value for the supernatant dilution. Using the standard curve from (8), the peptide concentration of the supernatant samples was determined and multiplied by their dilution factors to calculate the liraglutide concentration in the undiluted supernatant. Samples having a calculated concentration in the range of about 250 - 1000 μg / mL are preferred (this is in the middle of the accurate range of the BCA assay). 10. Since the fibrils aggregated at a concentration of 6 mg / mL (6000 μg / mL), the peptide concentration of the supernatant was subtracted therefrom to obtain the concentration of fibrils in the resuspended sample. For this calculation to be accurate, it is important to resuspend the fibril pellet of Assay (I) in exactly the same total volume after centrifugation.

[0076] Similar procedures may be used for semaglutide fibrils, except that references to Assay (I) in this specification should be replaced by references to Assay (II) in this specification.

[0077] Results [Example 1][Antibody] Antibodies (scFv-Fc) having the amino acid sequences listed in Table 1 were prepared by recombinant expression and purified.

[0078] Table 1 lists the complete sequences of each antibody, with the text in bold indicating the positions of the CDRs (shown in the order of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3), and the CDRs were defined according to the Kabat antibody numbering scheme. Table 2 lists the amino acid sequences of the variable regions (V L sequences) of the light chains and the variable regions (V H sequences) of the heavy chains of the antibodies in Table 1. Table 3 lists the CDRs of the antibodies in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 2-1

Table 2-2

Table 3-1

Table 3-2

[0079] [Example 2][Specificity of Antibody to Liraglutide Fibrils] The antibodies of Example 1 were individually tested for their ability to bind to liraglutide fibrils as compared to background and / or soluble liraglutide. The tests were conducted according to Assay (III) or Assay (IV) as defined herein. The results are shown in Tables 4 and 5.

Table 4

Table 5

[0080] The results in Tables 4 and 5 show that the antibodies tested bind significantly more to liraglutide fibrils than soluble liraglutide. The results in Table 4 also show that the antibodies tested bind significantly more to liraglutide fibrils than background. The results in Table 5 show that the antibodies tested can also bind significantly more to liraglutide fibrils when tested in a mixture of high concentrations of soluble liraglutide.

[0081] [Example 3][Detection Sensitivity of Liraglutide Fibril - Antibody Compared to ThT Assay] The ThT assay was tested for its ability to bind to liraglutide fibrils in a mixture with excess soluble liraglutide, enabling comparison of the sensitivity with the antibodies of the present invention. Experiments were conducted according to Assay (VI) herein. The results are shown in Table 6. [Table 6]

[0082] [Example 4][Concentration-Dependent Binding Analysis of Liraglutide Antibodies] Antibodies were prepared as described in the "Antibody Expression and Purification" section. Antibodies E, M, and N were purified in two steps with a yield of over 20 mg / L. The purified antibodies were mainly monomers, as demonstrated by analytical size exclusion chromatography (over 95% monomers for E, M, and N, Figure 1). The sensitivity of the assay using the two-step purified antibodies (over 95% monomers) was also enhanced by the removal of BSA during the primary antibody incubation. These changes led to an improvement in Assay (III-B).

[0083] Antibodies E, M, and N of Example 1 were purified in two steps (over 95% monomers) and individually tested for their ability to bind to liraglutide fibrils compared to background and / or soluble liraglutide. Tests were conducted according to Assay ((III-B) herein. The results are shown in Table 7 (raw (not background-subtracted) antibody binding signals for aggregated and soluble liraglutide), as well as Table 8 (liraglutide fibril specificity of the antibodies (liraglutide fibril / monomeric liraglutide)). Three independent experiments were performed, and the reported values are the averages. [Table 7] [Table 8]

[0084] The results in Tables 7 and 8 show that the tested antibodies bind significantly more to liraglutide fibrils than soluble liraglutide.

[0085] It is easier to control the amount of antibody aggregates in different batches of the two-step purified antibody (more than 95% monomer), so using a highly purified antibody (more than 95% monomer) in the assay has the advantage of making Assay (III-B) more reproducible than the previous Assay (III) that used a one-step purified antibody (more than 5% antibody aggregates). It was also observed that since antibody aggregates contributed to the binding to liraglutide fibrils, removal of antibody aggregates using size exclusion chromatography decreased the antibody sensitivity at low antibody concentrations. However, the increase in antibody purity allowed the use of higher antibody concentrations due to a lower background signal, which enabled an improvement in assay sensitivity.

[0086] [Example 5][Detection Sensitivity of Liraglutide Fibril - Antibody Compared with ThT Assay] Antibodies were prepared as described in the "Antibody Expression and Purification" section. Antibodies E, M, and N were purified in two steps with a yield of more than 20 mg / L. The purified antibodies were mainly monomers as demonstrated by analytical size exclusion chromatography (more than 95% monomers for E, M, and N, Figure 1). The sensitivity of the assay using the two-step purified antibody (more than 95% monomer) was also enhanced by the removal of BSA during the primary antibody incubation and an increase in antibody concentration (from 5 to 50 nM), resulting in an improvement in Assay (VI-B).

[0087] The ThT assay was tested for its ability to bind to liraglutide fibrils in a mixture with excess soluble liraglutide to enable a comparison of sensitivity with antibody M of the present invention. Experiments were conducted according to Assay (VI-B) herein. The results are shown in Table 9.

Table 9

[0088] The results in Table 9 show that the sensitivity of the antibodies of the present invention for detecting liraglutide fibrils is orders of magnitude higher than that of the ThT assay. Furthermore, it was also found that the antibodies of the present invention detected liraglutide fibrils at concentrations of fibrils where the signal was not recorded in the ThT assay.

[0089] Since it is easier to control the amount of antibody aggregates in different batches of the two-step purified antibody (more than 95% monomer), using a highly purified antibody (more than 95% monomer) in the assay has the advantage of making the assay (VI-B) more reproducible than the previous assay (VI) using a one-step purified antibody (more than 5% antibody aggregates). It was also observed that since antibody aggregates contributed to the binding to liraglutide fibrils, removal of antibody aggregates using size exclusion chromatography decreased the antibody sensitivity at low antibody concentrations (e.g., 5 nM). However, the increase in antibody purity allowed the use of a higher antibody concentration (50 nM instead of 5 nM) due to a lower background signal, which enabled an improvement in assay sensitivity.

[0090] [Example 6][Reproducibility of Antibodies with More than 95% Monomer Purity] The antibody M of Example 1 was purified in two steps (more than 95% monomer) and tested for its ability to bind to liraglutide fibrils in the presence of excess soluble liraglutide, compared to soluble liraglutide. The test was conducted according to the assay (VI-B) herein. Two different batches of the antibody were tested, and a total of 4 independent experiments were carried out. The results are shown in Table 10. [Table 10]

[0091] The results in Table 10 show that using a highly purified antibody (more than 95% monomer) in the assay gives very reproducible results.

[0092] While certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Accordingly, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the present invention.

Claims

**Claim 1** An antibody that binds to liraglutide fibrils, wherein the antibody comprises H - CDR1, H - CDR2, and H - CDR3 of the variable region of the heavy chain, and L - CDR1, L - CDR2, and L - CDR3 of the variable region of the light chain, and the H - CDR1, H - CDR2, and H - CDR3, and L - CDR1, L - CDR2, and L - CDR3 are respectively a. SEQ ID NO: 39, 38, and 37, and SEQ ID NO: 42, 41, and 40, b. SEQ ID NO: 45, 44, and 43, and SEQ ID NO: 48, 47, and 46, c. SEQ ID NO: 51, 50, and 49, and SEQ ID NO: 54, 53, and 52, d. SEQ ID NO: 57, 56, and 55, and SEQ ID NO: 60, 59, and 58, e. SEQ ID NO: 63, 62, and 61, and SEQ ID NO: 66, 65, and 64, f. SEQ ID NO: 69, 68, and 67, and SEQ ID NO: 72, 71, and 70, g. SEQ ID NO: 75, 74, and 73, and SEQ ID NO: 78, 77, and 76, h. SEQ ID NO: 81, 80, and 79, and SEQ ID NO: 84, 83, and 82, i. SEQ ID NO: 87, 86, and 85, and SEQ ID NO: 90, 89, and 88, j. SEQ ID NO: 93, 92, and 91, and SEQ ID NO: 96, 95, and 94, k. SEQ ID NO: 99, 98, and 97, and SEQ ID NO: 102, 101, and 100, l. SEQ ID NO: 105, 104, and 103, and SEQ ID NO: 108, 107, and 106, m. SEQ ID NO: 117, 116, and 115, and SEQ ID NO: 120, 119, and 118, or n. SEQ ID NO: 123, 122, 121, and SEQ ID NO: 126, 125, and 124, corresponding antibody. **Claim 2** The antibody according to claim 1, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 109, and 110, or any of the sequences having a maximum of 20 amino acid substitutions, deletions, or insertions. **Claim 3** The antibody according to claim 1 or 2, wherein the antibody has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 109, and 110. **Claim 4** The antibody according to any one of claims 1 to 3, wherein the antibody is a single-chain Fv fragment or a single-chain Fv fragment further comprising an Fc domain.

5. Use of the antibody according to any one of claims 1 to 4 as an affinity ligand for the identification of liraglutide fibrils and / or for the removal of liraglutide fibrils from a mixture comprising liraglutide fibrils and soluble liraglutide.

6. A method for identifying and / or quantifying liraglutide fibrils, the method comprising: a) binding the antibody according to any one of claims 1 to 4 to liraglutide and the method further comprising: b) detecting the antibody bound to liraglutide fibrils and / or c) quantifying the antibody bound to liraglutide fibrils The method may or may not include these steps.

7. An assay for detecting liraglutide fibrils in excess of soluble and / or monomeric liraglutide, the assay comprising the antibody according to any one of claims 1 to 4.

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