Antibodies directed against liraglutide and use thereof

Abstract

To provide means for identifying aggregates (fibrils) of human GLP-1 (7-37) and analogues thereof with higher sensitivity.SOLUTION: The present invention relates to an antibody that binds to liraglutide fibrils prepared according to assay (I) described herein.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is 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 technology]

[0003] Human GLP-1(7-37) and its analogs are known to have a tendency to form various types of aggregates in solution. Certain types of such aggregates, referred to herein as fibrils, form irreversibly and are believed to be kept to a minimum in pharmaceutical preparations for administration to patients in liquid form. To date, the preferred method for assaying (i.e., identifying and / or quantifying) such fibrils is based on thioflavin T (ThT), a fluorophore that changes its emission spectrum upon binding 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, which is undesirable and time-consuming. A means of identifying such peptide fibrils with greater sensitivity, even in mixtures containing soluble forms of the peptide, is desirable. Summary of the Invention

[0004] In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, the fibrils prepared according to Assay (I) herein. In some embodiments, the invention relates to antibodies that bind to semaglutide fibrils, the fibrils optionally prepared according to Assay (II) herein. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, the fibrils optionally prepared according to Assay (I) herein, the antibodies having a level of binding to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, the level of binding being determined according to Assay (III) at a liraglutide fibril concentration of at least 25 μM. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, optionally prepared according to assay (I) herein, and having a limit of detection for liraglutide fibrils at a concentration at least 10-fold lower than the limit of detection for liraglutide fibrils in a ThT assay, the limit of detection being determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, optionally prepared according to assay (I) herein, and having a level of binding to liraglutide fibrils that is at least 5-fold higher than the binding level of the antibody to soluble liraglutide, the antibody having a monomeric purity greater than 95%, and the level of binding being determined according to assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, the fibrils optionally prepared according to assay (I) herein, wherein the antibody has a detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils in a ThT assay, the antibody having a monomeric 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 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 selected from the group consisting of SEQ ID NOs: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the 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 selected from the group consisting of SEQ ID NOs: 115 and 121, or any of such sequences with 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 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 heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of sequences 115, 116, and 117; sequence numbers 121, 122, 123; 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; 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 sequences 118, 119, and 120; sequence numbers 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 invention relates to an antibody that binds to liraglutide fibrils, the antibody comprising a heavy chain variable region according to any one of the preceding embodiments and a light chain variable region as defined herein. In some embodiments, the 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 such sequences with up to 20, such as up to 15 or up to 10, amino acid substitutions, deletions, or insertions. In some embodiments, the 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 such sequences with up to 20, such as up to 15 or up to 10, amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies have at least 80%, such as at least 90% or at least 95%, sequence identity 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 invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies have at least 80%, such as at least 90% or at least 95%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 109 and 110.

[0007] In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise 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 such sequences with up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise 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 such sequences with up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10.

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

[0009] In some embodiments, the invention relates to the use of an antibody as defined herein for the purification of liraglutide or semaglutide by removal or reduction of fibrils of liraglutide or semaglutide, by immobilizing the antibody on a solid surface, for example a chromatographic or membrane surface creating an affinity surface, and exposing a mixture containing both fibrils and soluble forms of liraglutide or semaglutide to the surface, resulting in isolation of the fibrils or portions thereof. [Brief explanation of the drawings]

[0010] [Figure 1] Analytical size exclusion chromatography of selected antibody variants, E, M, and N, is shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to antibodies that specifically bind to 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. Liraglutide or semaglutide fibrils are undesirable in pharmaceuticals. Therefore, the antibodies of the present invention enable the differentiation of liraglutide or semaglutide fibrils from their soluble forms. Such antibodies of the present invention have several technical advantages, including the identification and / or quantification of such fibrils, optionally in mixtures with their soluble forms, and providing a means for ensuring the sufficient 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 mixtures of soluble liraglutide. Such isolation may be performed by immobilization on a solid surface, such as a chromatographic column, a filter, or a membrane. In some embodiments, the antibodies of the invention enable a highly sensitive assay for detecting extremely low levels of peptide fibrils, optionally in the presence of a large excess of soluble forms of the peptide. In some embodiments, the terms "fibril", "peptide fibril", also in reference to the specific peptides liraglutide or semaglutide, refer to the type of aggregates that can be obtained according to Assay (I) herein for liraglutide or Assay (II) herein for semaglutide, and such fibrils appear in the form of thin threads, for example using transmission electron microscopy.

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

[0013] In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, the fibrils being prepared according to assay (I) herein. In some embodiments, the invention relates to antibodies that bind to semaglutide fibrils. In some embodiments, the invention relates to antibodies that bind to semaglutide fibrils, the fibrils being 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 for liraglutide fibrils in a ThT assay, the detection limit being optionally determined according to assay (VI) herein. In some embodiments, the antibody has 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. 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 for semaglutide fibrils in a ThT assay, where the detection limit is optionally determined according to assay (VI) herein. In some embodiments, 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. 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 antibodies that bind to liraglutide fibrils, optionally prepared according to Assay (I) herein, wherein the antibodies have a level of binding to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibodies to soluble liraglutide, wherein the level of binding is determined according to Assay (III) at a liraglutide fibril concentration of at least 25 μM. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, optionally prepared according to Assay (I) herein, wherein the antibodies have a limit of detection for liraglutide fibrils at a concentration that is at least 10-fold lower than the limit of detection for liraglutide fibrils in a ThT assay, wherein the limit of detection is determined according to Assay (VI) herein at a liraglutide fibril concentration of at least 1 μM. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, optionally prepared according to Assay (I) herein, wherein the antibody has a level of binding to liraglutide fibrils that is at least 5-fold higher than the antibody's binding level to soluble liraglutide, wherein the antibody has greater than 95% monomeric purity, and wherein the level of binding 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 antibodies that bind to liraglutide fibrils, optionally prepared according to Assay (I) herein, wherein the antibody has a limit of detection for liraglutide fibrils at a concentration at least 10-fold lower than the limit of detection for liraglutide fibrils in a ThT assay, wherein the antibody has greater than 95% monomeric purity, and wherein the limit of detection 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 antibodies that bind to liraglutide fibrils, and that are capable of detecting liraglutide fibrils at concentrations of 1 to 1000 ppm of fibrils in solution, such as 1 to 10 ppm of fibrils, alternatively 10 to 100 ppm of fibrils, or alternatively 100 to 1000 ppm of fibrils.

[0016] In some embodiments, the 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 selected from the group consisting of SEQ ID NOs: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the 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 selected from the group consisting of SEQ ID NOs: 115 and 121, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the 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 selected from the group consisting of SEQ ID NOs: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR3 sequence of SEQ ID NO: 37, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR3 sequence of SEQ ID NO: 43, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR3 sequence of SEQ ID NO: 49, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 55, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 61, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 67, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 73, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 79, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 85, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 91, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 97, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 103, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 115, or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the CDR3 sequence of SEQ ID NO: 121, or any of such sequences with 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 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 heavy chain of the antibody comprises CDR1, CDR2, and / or CDR3 sequences selected from the group consisting of sequences 115, 116, and 117; sequence numbers 121, 122, 123; 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 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 the sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the 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 such sequences with 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 such sequences with 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 such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 49, 50, and 51; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 55, 56, and 57; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 61, 62, and 63; or any of such sequences with 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 these sequences having one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 73, 74, and 75; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 79, 80, and 81; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 85, 86, and 87; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 91, 92, and 93; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 97, 98, and 99; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the variable region of the heavy chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 103, 104, and 105; or any of such sequences with 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 these 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 these sequences with 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 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 such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the 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 such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 54; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 58, 59, and 60; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 64, 65, and 66; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 70, 71, and 72; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 76, 77, and 78; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 82, 83, and 84; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 88, 89, and 90; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions.In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 94, 95, and 96; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 100, 101, and 102; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 106, 107, and 108; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 118, 119, and 120; or any of such sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody comprises a CDR1, CDR2, and / or CDR3 sequence selected from the group consisting of SEQ ID NOs: 124, 125, and 126; or any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. In some embodiments, the 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 according to any one of the preceding embodiments.In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise 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 such sequences with up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise a sequence selected from the group consisting of SEQ ID NOs: 109 and 110, or any of such sequences with up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies have at least 80%, such as at least 90% or at least 95%, sequence identity 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, wherein the antibody has 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 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 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 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 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 defined herein.

[0019] In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise 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 such sequences with up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibodies comprise 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 such sequences with 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, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 70%, alternatively greater than 75%, alternatively greater than 80%, alternatively greater than 85%, alternatively greater than 90%, alternatively greater than 95%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 70%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 75%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 80%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 85%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 90%. In some embodiments, antibodies that bind to liraglutide fibrils have a monomeric purity of greater than 95%. In some embodiments, the purity of antibodies that bind to liraglutide fibrils is determined by the method described herein under "Size Exclusion Chromatography" followed by the AUC 280nm The area under the curve (AUC) based on the absorbance at 280 nm of the peak of the monomeric antibody associated with the sum of 280nm ) will be determined in accordance with the decision of

[0022] Liraglutide and semaglutide Liraglutide and semaglutide are analogs of human GLP-1 (7-37) that contain a covalent bond moiety. The antibodies of the present invention bind to liraglutide fibrils and / or semaglutide fibrils. The term "fibrils" 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 antibodies of the present invention bind to liraglutide fibrils. In some embodiments, the antibodies of the present invention bind 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. Example 37 of WO98 / 08871 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, e.g., under the trademark Victoza®.

[0024] Semaglutide is N-ε26-[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. Example 4 of WO2006 / 097537 is incorporated herein by reference. Semaglutide fibrils can be prepared as described in Assay (II) herein. An example of soluble semaglutide is the commercially available solution manufactured by Novo Nordisk A / S, Denmark, for example under the trademark Ozempic®.

[0025] antibody In some embodiments, the present invention relates to one or more of a series of antibodies characterized by antibody functionality and / or CDRs, heavy chain variable region, light chain variable region amino acid sequences, and / or Fc domain sequences. In some embodiments, the term "CDR" as used herein 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 present invention relates to one or more of a series of antibodies characterized by antibody functionality and / or H-CDR3 amino acid sequences. In some embodiments, the present invention relates to one or more of a series of antibodies characterized by antibody functionality and / or CDR amino acid sequences (CDR1, CDR2, and CDR3 of the heavy chain variable region may be referred to herein as H-CDR1, H-CDR2, and H-CDR3. Similarly, CDR1, CDR2, and CDR3 of the light chain variable region may be referred to herein as L-CDR1, L-CDR2, and L-CDR3). In some embodiments, the present invention relates to one or more of a series of antibodies characterized by antibody functionality and / or amino acid sequences of the heavy chain variable region and light chain variable region. In some embodiments, the present invention relates to one or more of a series of antibodies characterized by antibody functionality and / or amino acid sequences of the heavy chain variable region, light chain variable region, and / or Fc domain sequence. In some embodiments, the antibody comprises 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 a heavy chain variable region and / or a light chain variable region.

[0026] The antibodies of the present invention may be in any format, including whole antibodies and antigen-binding fragments (ie, "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, an scFv or scFv-Fc antibody comprises a variable region of the heavy chain (V H ) and the light chain variable region (V L ), and scFv-Fc antibodies further comprise an Fc domain.

[0028] In some embodiments, the antibody is a full-length antibody, comprising standard antibody domains and regions, e.g., as described herein. A full-length antibody (or whole antibody) comprises four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (C H Each light chain contains a light chain variable region (V L ) and the light chain constant region (C L The heavy chain constant region contains three domains: C H 1. C H 2, and C H Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one domain, C L Includes.

[0029] The variable region of the heavy chain and the variable region of the light chain each contain a binding domain that interacts with an antigen. H and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lmay comprise three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region of an 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 (Clq) of the classical complement system.

[0030] In some embodiments, the antibody is an antibody fragment, and such fragments can be obtained using conventional recombinant or protein engineering techniques. Antibody fragments of the invention can be generated by truncation, for example, by removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Fragments can also be generated by one or more internal deletions. In some embodiments, antibodies of the invention are or comprise a fragment of any one of the antibodies described herein. In some embodiments, antibodies of the invention are or comprise an antigen-binding portion of one of the antibodies described herein, or a variant thereof. For example, antibodies of the invention can be a Fab fragment of one of the antibodies described herein, or a variant thereof, or an antibody of the invention can be a single-chain antibody derived from one of the antibodies described herein, or a variant thereof. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, Fv (typically a single-arm V of an antibody), and the like. L and V H ), 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 dAbs (typically V H ) fragment;V H , V L , VhH, and V-NAR; single V H and a single V LThese include monovalent molecules containing chains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, for example, 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 be associated or linked together to form functional antibody fragments. Various types of antibody fragments are described or discussed, for example, in Holliger and Hudson, Nat Biotechnol 2005;2S:1126-1136, WO2005 / 040219, and U.S. Patent Application Publication Nos. 2005 / 0238646 and 2002 / 0161201.

[0031] The term "complementarity-determining region" ("CDR") or "hypervariable region" as used herein refers to the amino acid residues of an antibody involved in antigen binding. CDRs generally consist of residues from CDR1, CDR2, and CDR3 in the light chain variable region and CDR1, CDR2, and CDR3 in the heavy chain variable region, and / or "hypervariable loops" as defined according to Kabat (Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed according to the method described in Kabat et al. (see above). As used herein, the term "Kabat" refers to the numbering system for the heavy and / or light chain variable regions as described, for example, in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment 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 shortening of, or insertion into, the framework regions (FRs) or CDRs of the variable regions. The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with a "standard" Kabat numbered sequence at the regions of homology. The term "framework region" or "FR" residues refers to those V regions that are not within the CDRs, as defined herein. H or V L Refers to amino acid residues. The fragment crystallizable region of an antibody (Fc domain) is the region of an antibody that can interact with cell surface receptors called Fc receptors, as well as some proteins of the complement system.

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

[0033] The term "antigen" can refer to the molecular entity used to generate an antibody. However, as used herein, the term "antigen" broadly refers to the target molecule that binds to or specifically binds to an antibody, and thus includes fragments or mimetics of the molecular entity used to generate the antibody. Antibodies can be generated by any method, including by immunization of animals or 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 fragment thereof and its corresponding antigen. Generally, "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 called 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 many different epitopes, which may include, but are not limited to, linear peptide antigenic determinants, conformational antigenic determinants consisting of one or more non-contiguous amino acids located close to each other in the native (mature) conformation, and post-translational antigenic determinants consisting of either all or part of a molecular structure covalently attached to the antigen, such as a carbohydrate group.

[0035] The terms "binding," "specific binding," and "specificity" of an antibody are used herein to describe the selectivity of an antibody or its antigen-binding fragment. An antibody according to the present invention can specifically bind to liraglutide fibrils or semaglutide fibrils, indicating that the antibody has a significantly lower binding level than other antigens. In some embodiments, significantly lower is a binding level that is at least 10-fold lower, such as at least 15-fold lower or at least 20-fold lower. The binding level can be determined according to assay (III) herein or assay (IV) herein. The binding level 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 as determined by the number of matches between strings of two or more amino acid residues, and may be determined as the percent exact match between the smaller of two or more sequences with gapped alignments (if any) processed by a particular mathematical model or computer program (i.e., "algorithm"). Sequence identity of polypeptides can be readily calculated by methods known in the art, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, 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 to determine sequence identity are designed to give the largest match between the sequences tested.Methods for determining sequence identity are described in publicly available computer programs. Preferred computer program methods for determining sequence identity between two sequences include the GCG program package, which includes 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 publicly available from 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 whose percent sequence identity is to be determined are aligned for optimal amino acid matching (the "match span" determined by the algorithm). A gap opening penalty (calculated as three times the average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used, and "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (usually a fraction (1 / 10) of the gap opening penalty), and 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 calculated using the following parameters, for example, 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 present invention contain 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 replacing one amino acid residue with another residue 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 amino acid groups: hydrophilic: Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; aliphatic: Val, Ile, Leu, Met; basic: Lys, Arg, His; aromatic: Phe, Tyr, Trp; and typically, any residue may be substituted with alanine.

[0038] In some embodiments, one or more unnatural amino acids are introduced by substitution or insertion into an antibody of the invention, including, but not limited to, D-isomers of the 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, citraline, 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 antibodies of the present invention can be prepared by introducing appropriate nucleotide changes into the nucleic acids of the present invention or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence. Combinations of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics. Variant (modified) polypeptides can be prepared using any technique known in the art. For example, polynucleotides 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 propagating the transformed bacteria for a suitable number of generations. Products derived from mutated / modified DNA can be easily screened using the techniques described herein to determine whether they retain 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 characteristics to be modified. Mutation sites can be modified individually or sequentially, for example, by (1) substituting conservative amino acid choices first, followed by more radical choices depending on the results achieved, (2) deleting the target residue, or (3) inserting another residue adjacent to the site where it was located. In some embodiments, amino acid sequence deletions range from about 1 to 15 residues, more preferably from about 1 to 10 residues, and typically from about 1 to 5 contiguous 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 other / 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 derived from different species, including mammalian species such as mouse, rat, rabbit, pig, or non-human primate. The antibody may be a rodent antibody, more particularly a murine antibody. Alternatively, the antibody may be derived 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 may be prepared according to methods known in the art, such as recombinant protein, cell culture, and immunological techniques. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel 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 Harbor Laboratory, (1988), and J.E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley and Sons (including all current editions), and other sources.

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

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

[0044] Antibody Methods and Uses In some embodiments, the present invention relates to the use of an antibody defined herein for identifying and / or quantifying liraglutide or semaglutide fibrils. In some embodiments, the present invention relates to the use of an antibody defined herein for isolating, including partially isolating, liraglutide or semaglutide fibrils from a solution containing soluble liraglutide or soluble semaglutide. Such identification and / or quantification can be performed by binding an antibody to the fibrils and subsequently detecting the bound antibody, for example, via enzyme-linked immunosorbent assay (ELISA). ELISA can be performed as known in the art. In some embodiments, an ELISA vessel (such as a microtiter plate) is first saturated. 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 comprises an Fc domain, the secondary antibody can bind to this Fc domain. If a marker is present on the secondary antibody, detection and / or quantification of the secondary antibody may be possible, such as a fluorophore that can be identified via spectroscopy. Quantification can be performed using fibril standards bound to the antibody of the present invention.

[0045] In some embodiments, the term "detection limit" as used herein refers to the lowest limit of detection, which is the lowest concentration of a substance that can be distinguished from its absence. In some embodiments, the term "detection limit" as used herein refers to a mixture / soluble specificity ratio of 3 determined according to assay (IV) herein. A comparison of detection limits using an antibody and a ThT assay can be performed according to assay (VI) herein. A comparison of detection limits using an antibody and a ThT assay can be performed according to assay (VI-B) herein. In some embodiments, the term "detection limit" as used herein with respect to an antibody refers to the detection limit of an assay using that antibody in an ELISA, such as assay (III) or assay (IV) herein. In some embodiments, the term "detection limit" as used herein with respect to an antibody refers to the detection limit of an assay using that antibody in an ELISA, such as assay (III-B) herein. In some embodiments, the term "detection limit" as used herein is three times the standard deviation of a control sample tested in duplicate, where 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 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 present invention relates to a method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the step of a) binding an antibody as defined herein to liraglutide fibrils or semaglutide fibrils.

[0049] In some embodiments, the invention relates to a method for quantifying liraglutide or semaglutide fibrils, the method comprising: a) binding an antibody as defined herein to the liraglutide or semaglutide fibrils; or the method of any one of the preceding embodiments, further comprising: b) detecting the antibody bound to the liraglutide or semaglutide fibrils. In some embodiments, the method further comprises: c) quantifying the antibody bound to the liraglutide or semaglutide fibrils, optionally by use of a standard of the fibrils. In some embodiments, the fibrils are in a solution. In some embodiments, the fibrils are in a solution further comprising soluble liraglutide. In some embodiments, the fibrils are in a solution further comprising no other peptides or proteins other than the liraglutide fibrils and, optionally, the soluble liraglutide.

[0050] In some embodiments, the method includes (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 antigen-binding fragments thereof under conditions such that the antibody binds to the one or more immobilized fibrils to form an antibody-fibril complex; and (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 includes (a) contacting a solid support containing a fibril-specific antibody with the sample, such that, if present in the sample, fibrils bind to the antibody and become immobilized on the surface to form a complex, and (b) detecting the complex.

[0052] Any solid support known in the art can be used in the methods described herein, including, but not limited to, solid supports made of polymeric materials in the form of planar substrates or beads. 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, such as microparticles. The terms "beads" and "particles" are used interchangeably herein to refer to substantially spherical solid supports. The terms "microparticles" and "microbeads" are used interchangeably herein to refer to microbeads or microparticles that occupy or are allowed to settle in an array of wells, such as an array of wells in a detection module. Various techniques known in the art can be used to attach proteins or peptides to solid supports, such as plates or microparticles. A wide range of techniques are known for attaching reactive moieties to proteins, such as the methods described in U.S. Pat. No. 5,620,850. Methods for attaching 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 volume of sample using any suitable method known in the art. As used herein, the term "contacting" refers to any type of mixing action that brings the solid support into sufficient proximity with one or more fibrils in a sample so that a binding interaction occurs if one or more fibrils are present in the sample. Contacting can be achieved in a variety of different ways, including mixing the sample with a multiwell plate or microparticles. Contacting can be repeated as many times as necessary. Incubation can be in a binding buffer that promotes specific binding interactions, such as albumin (e.g., BSA), non-ionic detergent (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). Other conditions for binding interactions, such as temperature and salt concentration, can also be determined empirically or based on manufacturer's instructions. For example, contacting 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 capable of generating a signal that is detectable by visual or instrumental means. A detectable label may be, for example, a signal generator such as a chromogen, a fluorescent compound, an enzyme, a chemiluminescent compound, a radioactive compound, or the like. In one embodiment, a detectable label may be a fluorescent compound such as a fluorophore. The presence or amount of fibrils in a sample may 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 present invention relates to an assay for detecting liraglutide fibrils over soluble liraglutide and / or monomeric liraglutide, comprising an antibody according to the present invention, which is capable of detecting liraglutide fibrils at a fibril concentration of 1 to 1000 ppm in solution, such as 1 to 10 ppm fibrils, alternatively 10 to 100 ppm fibrils, or alternatively 100 to 1000 ppm 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 provided in the singular also include plural references.

[0056] MODE FOR CARRYING OUT THE INVENTION Non-limiting embodiments of the present invention include the following. 1.Antibodies that bind to liraglutide fibrils. 2. An antibody that binds to liraglutide fibrils, wherein the fibrils are prepared according to assay (I) of the present specification. 3.Antibodies that bind to semaglutide fibrils. 4. An antibody that binds to semaglutide fibrils, wherein the fibrils are prepared according to assay (II) herein. 5. The antibody of 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 for liraglutide fibrils in a ThT assay, the detection limit being optionally determined according to assay (VI) herein. 6. The antibody of embodiment 1 or 2, wherein the antibody has 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. 7. The antibody of 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 for liraglutide fibrils in a ThT assay, the detection limit being optionally determined according to assay (VI-B) herein. 8. The antibody of 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. The antibody of embodiment 3 or 4, wherein the antibody has a limit of detection of semaglutide fibrils at a concentration at least 10 times lower, such as at least 100 times or at least 1000 times lower than the limit of detection of semaglutide fibrils in a ThT assay, the limit of detection being optionally determined according to assay (VI) herein. 10. The antibody of embodiment 3 or 4, wherein the antibody has a level of binding 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. The antibody of embodiment 6 or 10, wherein the level of binding is determined according to assay (IV) herein. 12. The antibody of embodiment 6 or 10, wherein the level of binding 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 selected from the group consisting of SEQ ID NOs: 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 is: a. SEQ ID NOs: 37, 38, and 39; b. SEQ ID NOs: 43, 44, and 45; c. SEQ ID NOs: 49, 50, and 51; d. SEQ ID NOs: 55, 56, and 57; e. SEQ ID NOs: 61, 62, and 63; f. SEQ ID NOs: 67, 68, and 69; g. SEQ ID NOs: 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 any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. 15. An antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the 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 any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. 16. An antibody that binds to liraglutide fibrils, the antibody comprising a heavy chain variable region according to any one of the preceding embodiments, and a light chain variable region according to 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 such 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, wherein the antibody has at least 80%, such as at least 90% or at least 95%, sequence identity 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 such 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 such sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 21. The antibody of any one of the preceding embodiments, wherein said antibody is an isolated antibody. 22. The antibody of any one of the preceding embodiments, wherein said antibody is an isolated antibody. 23. The antibody of any one of the preceding embodiments, wherein the antibody comprises an Fc domain. 24. The antibody of any one of the preceding embodiments, wherein the antibody is a single chain Fv fragment further comprising an Fc domain. 25. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. 26. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils. 27. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the semaglutide fibrils. 28. Use of the antibody of any one of the preceding embodiments for the identification of liraglutide 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, comprising the step of: a) binding an antibody according to any one of the preceding embodiments to liraglutide fibrils or semaglutide fibrils. 31. A method for quantifying liraglutide or semaglutide fibrils, comprising the step of: a) binding an antibody according to any one of the preceding embodiments to liraglutide or semaglutide fibrils. 32.b) The method of any one of the preceding embodiments, further comprising the step of detecting antibodies bound to liraglutide fibrils or semaglutide fibrils. 33.c) The method of any one of the preceding embodiments, further comprising the step of quantifying antibodies bound to liraglutide fibrils or semaglutide fibrils, optionally by use of standards of said fibrils. 34. The method of any one of the preceding embodiments, wherein the fibrils are in solution. 35. The method of any one of the preceding embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide. 36. The method of 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: a. a level of binding 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 level of binding of the antibody to soluble liraglutide; and / or b. An antibody having 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 for liraglutide fibrils in a ThT assay, said detection limit being arbitrarily determined according to assay (VI) of the present specification. 38. An antibody that binds to semaglutide fibrils, wherein the fibrils are optionally prepared according to assay (II) herein, and the antibody: c. a level of binding 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 level of binding of the antibody to soluble semaglutide; and / or d. An antibody having a detection limit for semaglutide 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 for semaglutide fibrils in a ThT assay, the detection limit being optionally determined according to assay (VI). 39. The antibody of embodiment 37 or 38, wherein the level of binding is determined according to assay (IV) herein. 40. The antibody of embodiment 37 or 38, wherein the level of binding 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 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 selected from the group consisting of SEQ ID NOs: 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 NOs: 37, 38, and 39; b. SEQ ID NOs: 43, 44, and 45; c. SEQ ID NOs: 49, 50, and 51; d. SEQ ID NOs: 55, 56, and 57; e. SEQ ID NOs: 61, 62, and 63; f. SEQ ID NOs: 67, 68, and 69; g. SEQ ID NOs: 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 any of said sequences with 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 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 any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. 45. An antibody that binds to liraglutide fibrils, the antibody comprising a heavy chain variable region of any one of the preceding embodiments, and a light chain variable region of any one of the preceding embodiments. 46. ​​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 such sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 47. An antibody that binds to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90% or at least 95%, sequence identity 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. 48. The antibody of any one of the preceding embodiments, wherein said antibody is an isolated antibody. 49. The antibody of any one of the preceding embodiments, wherein the antibody is a single chain Fv fragment. 50. The antibody of any one of the preceding embodiments, wherein the antibody comprises an Fc domain. 51. The antibody of any one of the preceding embodiments, wherein the antibody is a single chain Fv fragment further comprising an Fc domain. 52. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. 53. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils. 54. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to the semaglutide fibrils. 55. Use of the antibody of any one of the preceding embodiments for the identification of liraglutide 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 the step of: a) binding an antibody according to any one of the preceding embodiments to liraglutide fibrils or semaglutide fibrils. 58. A method for quantifying liraglutide or semaglutide fibrils, comprising the step of: a) binding an antibody according to any one of the preceding embodiments to liraglutide or semaglutide fibrils. 59.b) The method of any one of the preceding embodiments, further comprising the step of detecting antibodies bound to liraglutide fibrils or semaglutide fibrils. 60.c) The method of any one of the preceding embodiments, further comprising the step of quantifying antibodies bound to liraglutide fibrils or semaglutide fibrils, optionally by use of standards of said fibrils. 61. The method of any one of the preceding embodiments, wherein the fibrils are in solution. 62. The method of any one of the preceding embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide. 63. The method of any one of the preceding embodiments, wherein the fibrils are in a solution that does not further comprise 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. SEQ ID NOs: 115, 116, and 117; n. SEQ ID NOs: 121, 122, 123, or any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. 65. An antibody that binds to liraglutide fibrils, wherein the variable region of the light chain of the antibody is: aa. SEQ ID NOs: 118, 119, and 120, bb. SEQ ID NOs: 124, 125, and 126; or any of said sequences with one, two, or three amino acid substitutions, deletions, or insertions. 66. An antibody that binds to liraglutide fibrils, the antibody comprising a heavy chain variable region according to any one of the preceding embodiments, and a light chain variable region according to any one of the preceding embodiments. 67. An antibody that binds to liraglutide fibrils, comprising a sequence selected from the group consisting of SEQ ID NOs: 109 and 110, or any of said sequences with 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 fibrils, wherein the antibody has at least 80%, such as at least 90% or at least 95%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 109 and 110. 69. 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 such 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, 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 such sequences having up to 20 amino acid substitutions, deletions, or insertions, such as up to 15 or up to 10. 71. The antibody of any one of embodiments 37 to 70, wherein the antibody is an isolated antibody. 72. The antibody of any one of embodiments 37 to 71, wherein the antibody is a single-chain Fv fragment. 73. The antibody of any one of embodiments 37 to 72, wherein the antibody comprises an Fc domain. 74. The antibody of any one of embodiments 37 to 73, wherein the antibody is a single chain Fv fragment further comprising an Fc domain. 75. An antibody described in any one of embodiments 37 to 74, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. 76. The antibody of any one of embodiments 37 to 75, wherein the antibody specifically binds to the liraglutide fibrils. 77. An antibody described in 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 is a. a level of binding to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, as determined according to Assay (III) at a liraglutide fibril concentration of at least 25 μM; and / or b. A limit of detection of liraglutide fibrils at a concentration at least 10-fold lower than the limit of detection of liraglutide fibrils in the ThT assay, the limit of detection being determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM, and / or c. a level of binding to liraglutide fibrils that is at least 5-fold higher than the antibody's binding to soluble liraglutide, wherein the antibody has greater than 95% monomeric purity and the binding level is determined according to assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM; and / or d. An antibody described in any one of embodiments 37 to 77, having a detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils in a ThT assay, wherein the antibody has a monomer purity of greater than 95%, and the detection limit is determined according to assay (VI-B) of the present specification at a liraglutide fibril concentration of at least 0.025 μM. 79. The antibody of 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 fibrils, alternatively 10 to 100 ppm fibrils, or alternatively 100 to 1000 ppm fibrils. 80. The antibody of any one of embodiments 37-79, wherein the antibody has a monomeric purity of greater than 70%, alternatively greater than 75%, alternatively greater than 80%, alternatively greater than 85%, alternatively greater than 90%, alternatively greater than 95%. 81. The antibody of any one of embodiments 37 to 80, wherein the antibody has a monomeric purity of greater than 95%. 82. Use of an antibody according to any one of embodiments 37 to 81 for the identification of liraglutide or semaglutide fibrils. 83. Use of the antibody of 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 the step of: a) binding an antibody described in any one of embodiments 37 to 81 to liraglutide fibrils or semaglutide fibrils. 85. A method for quantifying liraglutide fibrils or semaglutide fibrils, comprising the step of: a) binding an antibody described in any one of embodiments 37 to 81 to liraglutide fibrils or semaglutide fibrils. 86.b) The method of embodiment 84 or 85, further comprising the step of detecting antibodies bound to liraglutide fibrils or semaglutide fibrils. 87.c) A method according to any one of embodiments 84 to 86, further comprising the step of quantifying antibodies bound to liraglutide fibrils or semaglutide fibrils, optionally by use of standards of said fibrils. 88. The method of any one of embodiments 84-87, wherein the fibrils are in solution. 89. The method of any one of embodiments 84-88, wherein the fibrils are in a solution further comprising soluble liraglutide. 90. The method of any one of embodiments 84-89, wherein the fibrils are in a solution that does not further comprise 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 the antibody of any one of embodiments 37-81, 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 fibrils, alternatively 10 to 100 ppm fibrils, or alternatively 100 to 1000 ppm fibrils. [Example]

[0057] List of Abbreviations PBS: Phosphate buffered saline (pH 7.4, aqueous solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) PES: Polyethersulfone scFv-Fc: single-chain variable fragment linked to an Fc domain ThT: Thioflavin T

[0058] material and method Antibody library preparation, selection, and cloning of selected antibody variants Antibodies were isolated through two stages of library selection. In the first stage of selection, a single-chain variable fragment (scFv) yeast surface display library was generated by utilizing the diversity of the heavy chain CDR3 (HCDR3) of 4D5 scFv. The scFv was genetically fused to the C-terminus of the yeast Aga2 protein via a flexible linker, enabling antibody display on the cell surface. The yeast-displayed antibody library was selected 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 The beads were first washed (2x) with 1 mL of sterile PBS. Soluble liraglutide (100 μg, from a 6 mg / mL stock in drug composition buffer) was diluted in PBS containing magnetic beads (final volume 800 μL) and coupled to the beads overnight (4°C without agitation). For beads coated with fibrillar liraglutide, 100 μg of liraglutide fibrils were coupled to beads in 800 μL of PBS overnight at room temperature with end-to-end mixing. The next day, the beads were washed (2x) with 1 mL of PBS supplemented with 10 mM glycine to quench any unreacted toluenesulfonyl groups on the beads, and then washed (2x) with 1 mL of PBS supplemented with 1 g / L BSA (PBS-B) before incubation with yeast. Eight rounds of positive selection were performed on beads coated with liraglutide fibrils in PBS-B supplemented with 1% milk. To isolate yeast with conformation-specific antibodies against liraglutide fibrils, the final three rounds of sorting incorporated negative selections performed against beads coated with soluble liraglutide in PBS-B prior to positive selection against liraglutide fibrils.

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

[0060] 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. Insertion of the scFv-encoding fragment was confirmed by Sanger sequencing. These plasmids express bivalent scFv-human Fc fusion proteins with a 6xHis tag and a 3xFLAG tag on the C-terminus of the antibody.

[0061] Antibody expression and purification Proteins were expressed using the Expi293F Expression System (catalog number A14635). Expi293F cells were subcultured and grown until they reached a viable cell density of approximately 3–5 million cells per mL. Plasmid (30 μg) was transfected into 25 mL of Expi293 cells. ExpiFectamine 293 and plasmid DNA complexes were prepared as described in the manufacturer's instructions. 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 transfection reagent and DNA complexes were incubated at room temperature for 20 minutes and then added to the Expi293 cells. The cells were incubated at 37°C and 5% CO2 with shaking. Enhancer 1 and 2 solutions were added to the cells 20 hours after transfection, according to the manufacturer's instructions. After 3 days, the medium containing the secreted antibody was collected and centrifuged at 3400 xg for 45 minutes to remove cells and associated 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, and then 0.5 mL of beads was added to 30 mL of clarified 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). The Protein A beads were then 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. The eluted antibody was then buffer-exchanged into PBS using Zeba Spin Desalting Columns (89891, Thermo Fisher Scientific). Protein concentrations were assayed via absorbance measurements at 280 nm (168,460–205,360 M -1 cm -1 (extinction coefficient of ).

[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 sodium, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 200 mM arginine. The column flow rate was 0.75 mL / min. The antibody sample (0.1 mg / mL) was injected (100 μL) onto the column (GE 28990944, Superdex 200 Increase 10 / 300 GL column, 10 mm inner diameter, 300 mm length), and the absorbance signal was monitored at 220 and 280 nm. For preparative SEC, a FRC-10A fraction collector was used to isolate the monomer fraction.

[0064] Assay (I): Preparation of liraglutide fibrils A 6 mg / mL liraglutide test solution 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), adjusted to a final pH of 8.15 using NaOH and / or HCl as needed, followed by syringe filtration (0.22 μm PES filter). Aliquots of 1 mL of liraglutide solution were 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] A positive ThT signal was used to monitor fibril formation by removing a small sample (approximately 75 μL) of the test solution from the tube and analyzing it according to assay (V) (ThT assay) described herein. If the sample showed fluorescence at least 5-fold higher than that of the freshly prepared test solution in assay (V) (ThT assay) herein, 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 (retained for analysis by assay (VII) (BCA assay) herein). The pellet was gently washed once with pH 8.15 drug composition buffer (without disturbing the pellet) and then resuspended in the original volume of pH 8.15 drug composition buffer (taking into account any volume removed for ThT analysis) and stored at 4 °C. The concentration of fibrils was determined according to assay (VII) herein. For this calculation to be accurate, it is important that the fibril pellet is resuspended in the exact same total volume after centrifugation.

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

[0067] A positive ThT signal was used to monitor fibril formation by removing a small sample (approximately 75 μL) of the test solution from the tube and analyzing it according to assay (V) (ThT assay) described herein. If the sample showed fluorescence at least 5 times higher than that of freshly prepared semaglutide in the pH 6.9 drug composition buffer in assay (V) (ThT assay), 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 (retained for analysis by assay (VII) (BCA assay)). The pellet was gently washed once with the pH 6.9 drug composition buffer (without disturbing the pellet) and then resuspended in the original volume of the pH 6.9 drug composition buffer (taking into account any volume removed for ThT analysis) and stored at 4 ° C. The concentration of fibrils was determined according to assay (VII) herein. For this calculation to be accurate, it is important that the fibril pellet be resuspended 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: Liraglutide fibrils prepared according to Assay (I) herein were resuspended in drug composition buffer (with a fibril concentration determined by Assay (VII) herein (BCA assay)), and approximately 300 μL of the sample was sonicated (3 cycles of 10 seconds on / 30 seconds off at 100% amplitude, FB-120 Sonic Dismembrator, Thermo Fisher Scientific) in a microcentrifuge tube on ice to obtain 25 μM liraglutide fibrils. The solution was diluted in PBS to 25 μM liraglutide fibrils, and 100 μL of the sample was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (Product No. 439454). b. For soluble liraglutide coated plates: 6 mg / mL liraglutide was solubilized in drug composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate), pH adjusted to 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 sample was dispensed into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). i. For the "background" plate: Dispense 100 μL of PBS into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). 2. The plate was covered with adhesive film, wrapped in aluminum foil and incubated overnight at 4°C. 3. The next day, the plates were washed three times by adding 300 μL of PBS to each well. 4. The plates were blocked by adding 300 μL of PBS supplemented with 0.1% Tween 20 and 10 g / L BSA to each well. The plates were then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 3 hours. 5. While the plate was blocking, the antibody samples were spun in a centrifuge at 21,000 x 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% Tween 20 and 1 g / L BSA. 6. Wash the plate three times by adding 300 μL of PBS to each well. 7. 100 μL of antibody solution was dispensed into each well. Each antibody was tested in duplicate (i.e., 2 wells per plate for each antibody). The plates were then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. 8. Secondary antibody solution was prepared by diluting 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% Tween 20 and 10 g / L BSA. 9. The plate was washed three times by adding 300 μL of PBS to each well. 10. 100 μL of secondary antibody solution was dispensed into each well. The plate was then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, the 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) was removed from the refrigerator, the solution was allowed to equilibrate to room temperature, and 2M (4N) H2SO4 was prepared. 11. The plate was 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 formed (5–10 min). 13. The reaction was quenched by the addition of 100 μL of 2M H2SO4. 14. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). Calculation: The ratio of ELISA signals (absorbance at 450 nm) was calculated for each antibody on the fibril-coated plate and its signal on the soluble liraglutide-coated plate and background plate. These ratios are the fibril / soluble ratio and fibril / background ratio reported herein. For example, if an antibody produces a signal of 1.5 for liraglutide fibrils, 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, then sterilized by passing it through a 0.22 μm PES filter using a 30 cc Luer-Lok 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 adhesive film, wrapped in 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 fibril-coated ELISA plates: Liraglutide fibrils prepared according to Assay (I) herein were resuspended in drug composition buffer (with a fibril concentration determined by Assay (VII) herein (BCA assay)), 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). The solution was diluted to 10 μM liraglutide fibrils in PBS, and 100 μL of the sample was dispensed into each well. b. For soluble liraglutide coated plates: 6 mg / mL liraglutide was solubilized in 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 sample was dispensed into each well. i. For the "background" plate: Dispense 100 μL of PBS into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454). 3. The plate was covered with adhesive film, wrapped in aluminum foil and incubated at room temperature for 3 hours without agitation. 4. During this 3 hour period, 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% Tween 20 and 1 g / L BSA (unless otherwise specified). 5. The plate was washed three times by adding 300 μL of PBS to each well. 6. 100 μL of antibody solution was dispensed into each well. The plate was then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. 7. Secondary antibody solution was prepared by diluting 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% Tween 20 and 10 g / L BSA. 8. The plate was washed three times by adding 300 μL of PBST (PBS supplemented with 0.1% Tween 20) to each well. 9. 100 μL of secondary antibody solution was dispensed into each well. The plate was then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, the 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) was removed from the refrigerator, the solution was allowed to equilibrate to room temperature, and 2M (4N) 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 formed (5–10 min). 12. The reaction was quenched by the addition of 100 μL of 2M H2SO4. 13. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). Calculation: The ratio of ELISA signals (absorbance at 450 nm) was calculated for each antibody on the fibril-coated plate and its signal on the soluble liraglutide-coated plate and background plate. These ratios are the fibril / soluble ratio and fibril / background ratio reported herein. For example, if an antibody produces a signal of 1.5 for liraglutide fibrils, 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.

[0070] Assay (IV): Antibody Specificity Ratio (Method 2) The antibody specificity ratio was determined as follows: The night before the assay: BSA was solubilized at 1 mg / mL in PBS, then sterilized by passing it through a 0.22 μm PES filter using a 30 cc Luer-Lok 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 adhesive film, wrapped in aluminum foil, and incubated overnight at 4°C.

[0071] Day of assay: 1. Liraglutide was solubilized at 60 mg / mL in 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 solution at pH 8.15 in approximately 300 μL of sample (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 a "fibril solution." 3. The sonicated fibril solution was diluted in soluble liraglutide solution to a final fibril concentration of 100 μM. The solution was then serially diluted further in soluble liraglutide solution to result in a sample with 0.1 μM fibrils. Two controls were used: i) PBS (no peptide), and ii) no fibrils (soluble liraglutide solution only). 4. The ELISA plate (coated with BSA) was removed from the refrigerator and the wells were washed 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 adhesive film, wrapped in aluminum foil, and incubated at room temperature for 3 hours without agitation. 6. During the above 3-hour incubation, approximately 75 μL of the antibody (e.g., scFv-Fc fusion protein) to be tested 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% Tween 20 + 1 g / L BSA and kept on ice until use. At the end of the 7.3 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 adhesive film, wrapped in 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% Tween 20 + 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 secondary antibody solution was added to each well. The plate was covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. 12. During the secondary antibody incubation, remove the 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) from the refrigerator and allow the solution to equilibrate to room temperature. Prepare 2M (4N) H2SO4. At the end of the 13.1 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 2M H2SO4. The absorbance at 16.450 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 the control wells with soluble liraglutide and no fibrils. This ratio is the mixture / soluble specificity ratio. For example, if a given antibody produces 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 peptide test solution 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 may be prepared according to assay (I) herein. Semaglutide fibrils may be prepared according to assay (II) herein. A 75 μL sample of the test solution was mixed with 1.36 μL of ThT stock solution (stock concentration: 2200 μM ThT) to reach a final ThT concentration of 40 μM in the peptide / ThT mixture. In the case of liraglutide, the final concentration in this mixture was 1571 μM liraglutide (calculated before fibrillation). 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 min, 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 antibody assay Detection of liraglutide fibrils in mixtures with soluble liraglutide was determined using the ThT detection method compared to an antibody assay. 1. Liraglutide was solubilized at 60 mg / mL in 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 solution at pH 8.15 in approximately 300 μL of sample (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 a "fibril solution." 3. The sonicated fibril solution was diluted in soluble liraglutide solution to a final fibril concentration of 100 μM. The solution was then serially diluted further in soluble liraglutide solution to result in samples of 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM fibrils. Two controls were used: i) PBS (no peptide), and ii) no fibrils (soluble liraglutide solution only). At this point, the solution obtained by diluting the fibril solution in the soluble liraglutide solution (from step 3) was added (100 μL) to the wells of the ovalbumin-coated ELISA plate and incubated for 2 hours at room temperature before ELISA detection using the antibodies of the present invention. The remainder of the ELISA protocol was carried out as described in Assay (IV) (steps 7 onwards). 4. The remaining samples (a mixture of fibrils and soluble liraglutide, as well as the PBS control and soluble liraglutide control from step 3) were incubated in microcentrifuge tubes at room temperature for 2.5 hours. 5. Thioflavin T (ThT) stock solution was prepared at a concentration of 2200 μM. ThT was added to the samples (initially 1600 μM total peptide concentration) to a final concentration of 40 μM, and 50 μL 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). ThT fluorescence (λex = 444 nm, λem = 482 nm) was measured for each sample using a Biotek Synergy Neo microplate reader. The final (total) peptide concentration in the peptide / ThT mixture was 1571 μM (calculated before fibrillation). 6. The fluorescence measurement of the solution containing fibrils was divided by the fluorescence measurement 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 to antibody assay Detection of liraglutide fibrils in mixtures with soluble liraglutide was determined using the ThT detection method compared to an antibody assay. 1. The night before the assay: BSA was solubilized at 1 mg / mL in PBS, then sterilized by passing it through a 0.22 μm PES filter using a 30 cc Luer-Lok 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 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 BSA-coated plates. a. Liraglutide was solubilized at 60 mg / mL in 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." b. Liraglutide fibrils (hereinafter, fibrils) obtained according to Assay (I) herein and resuspended in a drug composition buffer solution at pH 8.15 in approximately 300 μL of sample (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." c. The sonicated fibril solution was diluted in soluble liraglutide solution to a final fibril concentration of 100 μM. The solution was then serially diluted further in soluble liraglutide solution to result in samples of 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM fibrils. Two controls were used: i) PBS (no peptide), and ii) no fibrils (soluble liraglutide solution only). d. The solution obtained by diluting the fibril solution in the soluble liraglutide solution was added (100 μL) to the wells of the BSA-coated ELISA plate and incubated at room temperature for 3 hours. 3. The remaining ELISA protocol was carried out as described for assay (VI) with some modifications. During this 3-hour period, 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% Tween 20 (PBST). b. The plate was washed three times by adding 300 μL of PBS to each well. c. 100 μL of antibody solution was dispensed into each well. The plate was then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. d. Secondary antibody solution was prepared by diluting 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% Tween 20 and 10 g / L BSA. e. The plate was washed three times by adding 300 μL of PBST to each well. f. 100 μL of secondary antibody solution was dispensed into each well. The plate was then covered with adhesive film, wrapped in aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, the 1-Step Ultra-TMB ELISA Substrate (Thermo Fisher Scientific, 34208) was removed from the refrigerator, the solution was allowed to equilibrate to room temperature, and 2M (4N) 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 formed (5–10 min). The reaction was quenched by the addition of i.100 μL of 2M H2SO4. j. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo). 4. The remaining samples (a mixture of fibrils and soluble liraglutide, as well as the PBS control and soluble liraglutide control from step 3) were incubated in microcentrifuge tubes at room temperature for 2.5 hours. 5. Thioflavin T (ThT) stock solution was prepared at a concentration of 2200 μM. ThT was added to the samples (initially 1600 μM total peptide concentration) to a final concentration of 0.4 μM, and 50 μL 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). ThT fluorescence (λex = 444 nm, λem = 482 nm) was measured for each sample using a Biotek Synergy Neo microplate reader. The final (total) peptide concentration in the peptide / ThT mixture was 1571 μM (calculated before fibrillation). 6. The fluorescence measurement of the solution containing fibrils was divided by the fluorescence measurement of the soluble liraglutide solution (control without fibrils) and the ratio was reported as the mixture / soluble ratio.

[0075] Assay (VII): BCA assay The concentration of fibrils (e.g., liraglutide fibrils) was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) with liraglutide as a liraglutide fibril standard and semaglutide as a semaglutide fibril standard (but not BSA). Because phenol from the drug composition buffer reacts with the BCA reagent to varying degrees depending on the sample dilution, a control run was performed for quantification to account for the background signal resulting from phenol. 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 in the fibril assembly (6 mg / mL). Analysis of liraglutide fibrils was performed as described below: 1. Liraglutide was dissolved at 6 mg / mL (6000 μg / mL) in drug composition buffer, pH 8.15. This is referred to as "soluble liraglutide solution." 2. For the standards, the soluble liraglutide solution from (1) was diluted 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, pH 8.15, diluted in PBS (but lacking peptide) was prepared. For example, to make 600 μg / L of the 2000 μg / mL standard requires 200 μL of the solution from (1) and 400 μL of PBS. To make the blank, 200 μL of drug composition buffer (without peptide) and 400 μL of PBS were mixed. 3. Supernatant from ultracentrifuged fibrils (in drug composition buffer, pH 8.15) obtained from assay (I) herein was diluted in PBS at the following dilutions: 1:2, 1:4, 1:8, 1:16, 1:32. "Blanks" were also prepared for these samples containing drug composition buffer, pH 8.15, diluted in PBS at the same dilutions. 4. To each well of a clear (no or low binding) flat-bottom 96-well plate, 10 μL of standard (and a separate well with the corresponding blank) and 10 μL of 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 adhesive film. 6. The plates were incubated at 37°C until sufficient purple color formation occurred (this generally occurred relatively quickly, and some color was often visible in some samples almost immediately). The absorbance was read on a plate reader at 7.562 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 peptide concentration versus absorbance and fitting a second-order polynomial. 9. The "blank" absorbance values ​​for the supernatant were subtracted from the values ​​for the supernatant dilutions. Using the standard curve from (8), the peptide concentrations of the supernatant samples were determined and multiplied by their dilution factors to calculate the liraglutide concentration in the undiluted supernatant. Samples with calculated concentrations in the range of approximately 250-1000 μg / mL are preferred (this is in the middle of the accurate range of the BCA assay). 10. Because fibrils were assembled at a concentration of 6 mg / mL (6000 μg / mL), the peptide concentration in the supernatant was subtracted from this to obtain the concentration of fibrils in the resuspended sample. For this calculation to be accurate, it is important to resuspend the fibril pellet from Assay (I) in the exact same total volume after centrifugation.

[0076] A similar procedure may be used for semaglutide fibrils, except that references to assay (I) herein should be replaced with assay (II) herein.

[0077] result [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 sequence of each antibody, with bold text indicating the CDR positions (shown in the order L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3), where the CDRs are defined according to the Kabat antibody numbering scheme. Table 2 lists the variable regions (V) of the light chains of the antibodies in Table 1. L sequence), heavy chain variable region (V H 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] [Antibody specificity to liraglutide fibrils] The antibodies of Example 1 were individually tested for their ability to bind to liraglutide fibrils, compared to background and / or soluble liraglutide. Testing was performed according to Assay (III) or Assay (IV) 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 tested antibodies bind significantly more to liraglutide fibrils than to soluble liraglutide. The results in Table 4 also show that the tested antibodies bind significantly more to liraglutide fibrils than background. The results in Table 5 show that the tested antibodies 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 with ThT assay] The ThT assay was tested for its ability to bind to liraglutide fibrils in a mixture with excess soluble liraglutide, allowing for a comparison of sensitivity with the antibodies of the present invention. The experiment was performed according to assay (VI) herein. The results are shown in Table 6. [Table 6]

[0082] [Example 4] [Concentration-dependent binding analysis of liraglutide antibody] Antibodies were prepared as described in the "Antibody Expression and Purification" section. Antibodies E, M, and N were two-step purified with yields of >20 mg / L. The purified antibodies were predominantly monomeric, as demonstrated by analytical size-exclusion chromatography (>95% monomeric for E, M, and N, Figure 1). The sensitivity of the assay using the two-step purified antibodies (>95% monomeric) was also enhanced by the removal of BSA during the primary antibody incubation. These changes resulted in an improvement of the assay (III-B).

[0083] Antibodies E, M, and N from Example 1 were two-step purified (>95% monomer) and individually tested for their ability to bind to liraglutide fibrils compared to background and / or soluble liraglutide. Testing was performed according to the assay (III-B) herein. The results are shown in Table 7 (raw (non-background subtracted) antibody binding signals for aggregated liraglutide and soluble liraglutide) and Table 8 (antibody liraglutide fibril specificity (liraglutide fibrils / monomeric liraglutide)). Three independent experiments were performed, and reported values ​​are averages. [Table 7] [Table 8]

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

[0085] It was also observed that using highly purified antibodies (>95% monomer) in the assay had the advantage of making assay (III-B) more reproducible than the previous assay (III) using single-step purified antibodies (>5% antibody aggregates), because it was easier to control the amount of antibody aggregates in different batches of two-step purified antibodies (>95% monomer). Removal of antibody aggregates using size exclusion chromatography reduced antibody sensitivity at low antibody concentrations because antibody aggregates contributed to binding to liraglutide fibrils. However, increased antibody purity allowed the use of higher antibody concentrations due to lower background signal, which improved 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 two-step purified with yields of >20 mg / L. The purified antibodies were predominantly monomeric, as demonstrated by analytical size-exclusion chromatography (>95% monomeric for E, M, and N, Figure 1). The sensitivity of the assay using the two-step purified antibodies (>95% monomeric) was also enhanced by removing BSA during the primary antibody incubation and increasing the antibody concentration (5 to 50 nM), resulting in an improved 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 allow for a comparison of sensitivity with antibody M of the present invention. The experiment was performed 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 several 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 no signal was recorded in the ThT assay.

[0089] It was also observed that using highly purified antibodies (>95% monomer) in the assay had the advantage of making assay (VI-B) more reproducible than the previous assay (VI) using single-step purified antibodies (>5% antibody aggregates), because it was easier to control the amount of antibody aggregates in different batches of two-step purified antibodies (>95% monomer). Removal of antibody aggregates using size exclusion chromatography reduced antibody sensitivity at low antibody concentrations (e.g., 5 nM) because antibody aggregates contributed to binding to liraglutide fibrils. However, increased antibody purity allowed the use of higher antibody concentrations (50 nM instead of 5 nM) due to lower background signal, which improved assay sensitivity.

[0090] Example 6 Reproducibility of antibodies with greater than 95% monomer purity Antibody M from Example 1 was two-step purified (>95% monomer) and tested for its ability to bind to liraglutide fibrils in the presence of excess soluble liraglutide compared to soluble liraglutide. Testing was performed according to assay (VI-B) herein. Two different batches of antibody were tested, for a total of four independent experiments. The results are shown in Table 10. [Table 10]

[0091] The results in Table 10 show that using highly purified antibodies (>95% monomeric) in the assay gives highly reproducible results.

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

Claims

1. An antibody that binds to liraglutide fibrils, said fibrils optionally prepared according to assay (I) herein, said antibody comprising: a. a level of binding to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, wherein the binding level is determined at a liraglutide fibril concentration of at least 25 μM according to assay (III), and / or b. A detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils of the ThT assay, said detection limit being determined according to assay (VI) herein at a liraglutide fibril concentration of at least 1 μM, and / or c. A level of binding to liraglutide fibrils that is at least 5-fold higher than the level of binding of the antibody to soluble liraglutide, wherein the antibody has a purity of greater than 95% free base content, and the level of binding is determined according to assay (III-B) herein at a liraglutide fibril concentration of at least 30 μM; and / or d. A detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils in a ThT assay, wherein the antibody has a purity of greater than 95% of the amount of free radicals, and the detection limit is determined according to assay (VI-B) herein at a liraglutide fibril concentration of at least 0.025 μM. An antibody having

2. The fibrils are optionally prepared according to assay (I) herein, and the antibody is a. a level of binding to liraglutide fibrils that is at least 10-fold higher than the binding level of the antibody to soluble liraglutide, wherein the level of binding is determined at a liraglutide fibril concentration of at least 25 μM according to assay (III), and / or b. The antibody of claim 1, having a detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils in a ThT assay, wherein the detection limit is determined at a liraglutide fibril concentration of at least 1 μM according to assay (VI) of the present specification.

3. The fibrils are optionally prepared according to assay (I) herein, and the antibody is c. a level of binding to liraglutide fibrils that is at least 5-fold higher than the level of binding of the antibody to soluble liraglutide, wherein the antibody has a purity of greater than 95% free base content, and the level of binding is determined at a liraglutide fibril concentration of at least 30 μM according to assay (III-B) herein; and / or d. The antibody of claim 1 or 2, having a detection limit for liraglutide fibrils at a concentration at least 10-fold lower than the detection limit for liraglutide fibrils in a ThT assay, wherein the antibody has a purity of greater than 95% of the monolayer amount, and the detection limit is determined according to assay (VI-B) herein at a liraglutide fibril concentration of at least 0.025 μM.

4. 4. The antibody of any one of claims 1 to 3, wherein the antibody has a purity by single unit amount of greater than 70%, alternatively greater than 75%, alternatively greater than 80%, alternatively greater than 85%, alternatively greater than 90%, alternatively greater than 95%.

5. 5. The antibody of any one of claims 1 to 4, wherein the antibody is capable of detecting liraglutide fibrils at a concentration of 1 to 1000 ppm of fibrils in solution, such as 1 to 10 ppm of fibrils, alternatively 10 to 100 ppm of fibrils, alternatively 100 to 1000 ppm of fibrils.

6. 6. The antibody of any one of claims 1 to 5, wherein the variable region of the heavy chain of the antibody comprises a CDR3 sequence 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 foregoing sequences having one, two or three amino acid substitutions, deletions or insertions.

7. The heavy chain variable region of the antibody is a. SEQ ID NOs: 37, 38, and 39; b. SEQ ID NOs: 43, 44, and 45; c. SEQ ID NOs: 49, 50, and 51; d. SEQ ID NOs: 55, 56, and 57; e. SEQ ID NOs: 61, 62, and 63; f. SEQ ID NOs: 67, 68, and 69; g. SEQ ID NOs: 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; m. SEQ ID NOs: 115, 116, and 117; n. SEQ ID NOs: 121, 122, 123, or any of the foregoing sequences having 1, 2, 3 amino acid substitutions, deletions, or insertions.

8. the variable region of the light chain of the antibody is SEQ ID NOs: 40, 41, and 42; p. SEQ ID NOs: 46, 47, and 48; q. SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 64, 65, and 66; t. SEQ ID NOs: 70, 71, and 72; SEQ ID NOs: 76, 77, and 78; v. SEQ ID NOs: 82, 83, and 84; w. SEQ ID NOs: 88, 89, and 90; x. SEQ ID NOs: 94, 95, and 96; y. SEQ ID NOs: 100, 101, and 102; z. SEQ ID NOs: 106, 107, and 108; aa. SEQ ID NOs: 118, 119, and 120; bb. SEQ ID NOs: 124, 125, and 126; or any of the foregoing sequences having one, two, three amino acid substitutions, deletions, or insertions.

9. The antibody of any one of claims 1 to 8, wherein the antibody comprises a heavy chain variable region as defined in claim 6 or 7 and a light chain variable region as defined in claim 8.

10. 10. The antibody of any one of claims 1 to 9, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 109, and 110, or any of said sequences having up to 20, such as up to 15 or up to 10 amino acid substitutions, deletions or insertions.

11. The antibody of any one of claims 1 to 10, wherein the antibody has at least 80%, such as at least 90% or at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 109 and 110.

12. The antibody of any one of claims 1 to 11, wherein the antibody is a single-chain Fv fragment or a single-chain Fv fragment further comprising an Fc domain.

13. Use of an antibody according to any one of claims 1 to 12 as an affinity ligand for identifying liraglutide fibrils and / or for removing fibrils from a mixture containing liraglutide fibrils and soluble substances.

14. 1. A method for identifying and / or quantifying liraglutide fibrils, said method comprising: a) binding the antibody according to any one of claims 1 to 12 to liraglutide; b) optionally detecting antibodies bound to liraglutide fibrils; c) optionally quantifying antibody bound to liraglutide fibrils, optionally by use of standards of said fibrils.

15. 13. An assay for detecting liraglutide fibrils over soluble and / or monomeric liraglutide, comprising the antibody of any one of claims 1 to 12, wherein the antibody is capable of detecting liraglutide fibrils at a concentration of 1 to 1000 ppm of fibrils in solution, such as 1 to 10 ppm of fibrils, alternatively 10 to 100 ppm of fibrils, or 100 to 1000 ppm of fibrils.

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