Elenumab compositions and uses thereof

Abstract

To provide an elenumab composition and its use.SOLUTION: The present invention relates to compositions comprising enumab and one or more enumab variants, including isomerized variants, deamidated variants, acidic variants, and HMW species. Pharmaceutical formulations comprising the enulumab compositions and methods of using and characterizing the compositions are also described. The present invention is based, in part, on the identification and characterization of product-related impurities of Enumab, in particular variants of Enumab with reduced functional activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 651,651, filed April 2, 2018, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on March 1, 2019, is titled A-2190-WO-PCT_SeqList_ST25 and is 15 kilobytes in size.

[0003] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to the identification and characterization of erenumab variants that affect the functional activity of erenumab. The present invention also relates to compositions comprising erenumab and one or more erenumab variants, pharmaceutical formulations comprising the compositions, and methods of using and characterizing the compositions. [Background technology]

[0004] Aimovig is a fully human monoclonal antibody that specifically binds to the human calcitonin gene-related peptide (CGRP) receptor and prevents CGRP ligands from binding to and activating the receptor. CGRP has been implicated in the pathogenesis of migraine (Durham, New England Journal of Medicine, Vol. 350:1073-1075, 2004; Edvinsson et al., Neurotherapeutics, Vol. 7:164-175, 2010). In phase 2 and 3 clinical trials, patients with chronic or episodic migraine treated with Aimovig experienced a reduction in the number of migraine days per month compared with patients receiving placebo (Tepper et al., Lancet Neurol., Vol. 16:425-434, 2017; Goadsby et al., New England Journal of Medicine, Vol. 377:2123-2132, 2017).

[0005] Complex therapeutic biologics, such as monoclonal antibodies, can have numerous product quality attributes that can affect the safety and / or efficacy of the biologic. Identifying such attributes and understanding their influence on the molecule's properties typically requires a comprehensive structural and functional evaluation of the molecule. Understanding the molecular attributes is important to ensure consistent product manufacturing.

[0006] Variants of biologics can arise during the manufacturing process or under storage conditions. Such product variants can be classified as product-related substances or product-related impurities. Product-related substances are molecular variants of the desired product formed during product manufacturing or storage that have properties comparable to those of the desired product and are not considered impurities. Conversely, product-related impurities are molecular variants of the desired product that do not have properties comparable to those of the desired product with respect to product efficacy and patient safety. Therefore, the identification and characterization of product-related impurities is particularly useful in the manufacturing and quality control of biologic drug products. Summary of the Invention [Means for solving the problem]

[0007] The present invention is based, in part, on the identification and characterization of product-related impurities of erenumab, particularly variants of erenumab with reduced functional activity. Controlling and monitoring the amount of these variants in therapeutic erenumab compositions ensures that the erenumab composition has the potency necessary to produce the desired clinical effect. Thus, in some embodiments, the present invention provides erenumab compositions comprising erenumab and one or more erenumab variants, wherein the erenumab variants include isomerized variants, desamidated variants, acidic variants, disulfide isoform variants, high molecular weight (HMW) species, or combinations thereof, and the amount of the erenumab variants in the composition is controlled within a specific range.

[0008] In certain embodiments, the invention provides compositions comprising Aimovig and one or more Aimovig variants, wherein the one or more Aimovig variants include isomerized and desamidated variants. In some embodiments, the isomerized variants have an isoaspartic acid residue or succinimide at amino acid position 105 in one or both heavy chains of Aimovig (SEQ ID NO: 1 or SEQ ID NO: 3). In these and other embodiments, the desamidated variants have an asparagine residue at amino acid position 102 in one or both heavy chains of Aimovig (SEQ ID NO: 1 or SEQ ID NO: 3) converted to an aspartic acid residue, succinimide, or isoaspartic acid residue. The amount of isomerized and desamidated variants in the composition can be less than about 30%, less than about 15%, less than about 8%, or less than about 4%, as determined, for example, by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

[0009] The present invention also provides compositions of erenumab and one or more acidic variants of erenumab. The amount of acidic variants in the composition can be less than about 40%, for example, about 25% to about 38%, or about 26% to about 34%. In some embodiments, the amount of acidic variants in the erenumab composition is determined by cation exchange high-performance liquid chromatography (CEX-HPLC). Acidic variants of erenumab include disulfide isoform variants, desamidated variants, fragmented variants, non-consensus glycosylation variants, HMW species, and combinations thereof. In certain embodiments, the acidic variants include disulfide isoform variants, fragmented variants, or combinations thereof.

[0010] In some embodiments, the present invention provides compositions of Aimovig and disulfide isoform variants of Aimovig. Aimovig disulfide isoform variants may include the IgG2-B isoform and / or the IgG2-A / B isoform. The amount of IgG2-B isoform in the composition may be less than about 20%, e.g., less than about 10%, or less than about 8%, e.g., about 4% to about 6%. The amount of IgG2-A / B isoform in the composition may be about 20% to about 40%, about 26% to about 38%, or about 34% to about 37%. In certain embodiments, the amount of disulfide isoforms is determined by non-reducing reverse-phase high-performance liquid chromatography (RP-HPLC).

[0011] The present invention also includes compositions comprising erenumab and one or more size variants of erenumab, such as low molecular weight (LMW) species, medium molecular weight (MMW) species, and high molecular weight (HMW) species. In certain embodiments, the amount of HMW species in the composition is less than about 3.0%, for example, about 2.5% or less, about 2.1% or less, about 1.8% or less, about 1.4% or less, or about 1.2% or less. In one specific embodiment, the HMW species of erenumab are mainly composed of covalently linked dimers of erenumab. The amount of HMW species in erenumab compositions can be determined by size exclusion ultra-high performance liquid chromatography (SE-UHPLC).

[0012] In some embodiments, the present invention also provides a method for evaluating or assessing the quality of an erenumab composition. In one embodiment, the method includes obtaining an erenumab composition containing erenumab and one or more erenumab variants; measuring the amount of one or more erenumab variants in the composition; comparing the measured amount of one or more erenumab variants to a predetermined reference standard; and, if the comparison indicates satisfaction of the predetermined reference standard, producing a pharmaceutical formulation or product of the erenumab composition. The method may include one, two, or three of the following: (1) measuring the amount of isomerized variants and deamidated variants in the composition (e.g., by the peak area percentage of the pre-peak in HIC-HPLC), (2) measuring the amount of acidic variants in the composition (e.g., by the peak area percentage of the acidic peak in CEX-HPLC), and / or (3) measuring the amount of HMW species in the composition (e.g., by the peak area percentage of the pre-peak in SE-UHPLC). In certain embodiments, all three measurements are performed on the erenumab composition.

[0013] Pharmaceutical preparations comprising the erenumab compositions described herein are also included in the present invention.In some embodiments, the pharmaceutical preparation comprises the erenumab compositions described herein and one or more pharmaceutically acceptable excipients, such as buffers, sugars, and surfactants.The pharmaceutical preparation can be incorporated into an injection device, such as a pre-filled syringe or an automatic injection device.In such embodiments, the injection volume for the pre-filled syringe or automatic injection device is less than about 2 mL, for example, about 1 mL.

[0014] The present invention also includes methods for treating, preventing, or reducing the incidence of headache in a patient in need thereof using the erenumab compositions and pharmaceutical formulations of the present invention. In one embodiment, the method comprises administering to the patient a pharmaceutical formulation comprising the erenumab composition described herein. The headache may be migraine, cluster headache, or other types of headache disorders, such as tension-type headache, hemiplegic migraine, menstrual-related migraine, and retinal migraine. In certain embodiments, the patient to be treated according to the method or use of the present invention has or has been diagnosed with migraine, such as episodic migraine or chronic migraine.

[0015] The use of erenumab compositions for the preparation of a medicament for administration in any of the methods described herein or by any of the methods disclosed herein is specifically contemplated. For example, the present invention includes erenumab compositions or pharmaceutical formulations described herein for use in a method for treating, preventing, or reducing the incidence of headache in a patient in need thereof. The present invention also encompasses the use of erenumab compositions or pharmaceutical formulations described herein in the preparation of a medicament for treating, preventing, or reducing the incidence of headache in a patient in need thereof. [Brief explanation of the drawings]

[0016] [Figure 1A-1B] The amino acid sequences of the heavy chain (FIG. 1A; SEQ ID NO: 1) and light chain (FIG. 1B; SEQ ID NO: 2) for Aimovig are shown. The complementarity-determining regions (CDRs) in each chain are bolded and underlined. The heavy chain contains an N-linked glycosylation site at asparagine residue 306. [Figure 1C-1D] The amino acid sequences of the heavy chain (FIG. 1C; SEQ ID NO: 3) and light chain (FIG. 1D; SEQ ID NO: 4) with common post-translational modifications at the N- and C-termini for Aimovig are shown. The CDRs in each chain are bolded and underlined. pE stands for pyroglutamic acid. [Figures 1E-1F]The amino acid sequences of the heavy chain variable region (FIG. 1E; SEQ ID NO: 5) and light chain variable region (FIG. 1F; SEQ ID NO: 6) for Aimovig are shown. The CDRs in each variable region are bolded and underlined. [Figure 2A-2B] Representative HIC-HPLC profiles of erenumab drug substance at full scale (Figure 2A) and expanded scale (Figure 2B) are shown. Erenumab drug substance was analyzed by HIC-HPLC using a mobile phase of sodium acetate pH 5.5 with elution with a linear decreasing gradient of ammonium sulfate and detection at 280 nm absorbance. [Figure 3] An overlay of HIC-HPLC collected fractions and Aimovig drug substance analyzed by HIC-HPLC is shown. Aimovig drug substance and the fractions collected from semi-preparative HIC-HPLC (F1-F7) were analyzed by HIC-HPLC using a mobile phase of sodium acetate pH 5.5 with elution with a linear decreasing gradient of ammonium sulfate and detection at 280 nm absorbance. Each panel is a plot of absorbance at 280 nm versus elution time in minutes. An overlay of all seven HIC-HPLC fractions with Aimovig drug substance is shown in panel 8. [Figure 4] Figure 1 shows an overlay of reduced tryptic peptide maps of the elution region for peptides within the heavy chain CDR3 of erenumab for enriched HIC-HPLC fractions (F2-F6) and unfractionated erenumab bulk drug (DS). The TH12H13 peptide corresponds to amino acid residues 99-113 of SEQ ID NO:1, and TH13 corresponds to amino acid residues 101-113 of SEQ ID NO:1. [Figure 5] 1 shows the HIC-HPLC profile of erenumab drug substance exposed to heat stress. Erenumab drug substance incubated at 50°C for 14 days was analyzed by HIC-HPLC using a mobile phase of sodium acetate pH 5.5 with elution with a linear gradient of ammonium sulfate and detection at 280 nm absorbance. The top trace corresponds to the 14-day time point. [Figure 6]Figure 1 shows an expanded-scale reduced tryptic peptide map profile for Aimovig erenumab drug substance stressed at 50°C for 14 days. The TH12H13 peptide corresponds to amino acid residues 99-113 of SEQ ID NO: 1, and TH13 corresponds to amino acid residues 101-113 of SEQ ID NO: 1. The TH26 peptide corresponds to amino acid residues 311-326 of SEQ ID NO: 1, with the detected fragment resulting from nonspecific cleavage between L315 and T316. [Figure 7] Figure 1 shows the HIC-HPLC profile of Aimovig erenumab drug substance stressed at pH 7.4 and 37°C. Aimovig erenumab drug substance diluted to 10 mg / mL in PBS, pH 7.4, and incubated at 37°C for 14 days was analyzed by HIC-HPLC using a sodium acetate, pH 5.5 mobile phase with elution by a linear ammonium sulfate gradient and detection at 280 nm absorbance. Control samples, along with physiological pH stressed samples, were diluted in formulation buffer, pH 5.2, and incubated at 37°C for 14 days. [Figure 8] Figure 1 shows the HIC-HPLC profile of Aimovig erenumab drug substance stressed at pH 8.0 and 25°C. Aimovig erenumab drug substance diluted to 10 mg / mL in Tris base, pH 8.0, and incubated at 25°C for 14 days was analyzed by HIC-HPLC using a sodium acetate, pH 5.5 mobile phase with elution with a linear ammonium sulfate gradient and detection at 280 nm absorbance. A control sample, along with the high pH stress condition sample, was diluted in formulation buffer, pH 5.2, and incubated at 25°C for 14 days. [Figure 9] 1 is a plot of the relative potency percentage of a lot of Aimovig as a function of the peak area percentage of the pre-peak in HIC-HPLC analysis of the same lot for four different storage conditions. A regression line fitted from a linear regression analysis of the data is shown for each temperature. For the 40°C condition, the slope of the regression line is statistically different from zero, indicating how the relative potency percentage decreases with increasing HIC-HPLC pre-peak area percentage. [Figure 10]The plot and fitted regression line for the data from the 40° C. storage condition is shown in Figure 9 with the fitted line extended to intersect the 70% relative potency threshold, which intersects the regression line at approximately 28.85% of the HIC-HPLC pre-peak area. [Figure 11]

[0039] Figure 1 shows a representative CEX-HPLC profile of erenumab drug substance. Erenumab drug substance was analyzed by CEX-HPLC using a mobile phase of sodium phosphate pH 6.6 with elution with a linear gradient of sodium chloride and detection at 280 nm absorbance. [Figure 12] Shown is an overlay of CEX-HPLC collected fractions and Aimovig ® drug substance analyzed by CEX-HPLC. Aimovig ® drug substance and fractions collected from semi-preparative CEX-HPLC (F1-F9) were analyzed by CEX-HPLC using a mobile phase of sodium phosphate pH 6.6 with elution with a linear gradient of sodium chloride and detection at 280 nm absorbance. Each panel is a plot of absorbance at 280 nm against elution time in minutes. [Figures 13A-13B] Figure 13A shows the RP-HPLC profiles of the acidic and main peak fractions of CEX-HPLC and the basic peak fraction of CEX-HPLC (Figure 13B). Erenumab drug substance (DS) and CEX-HPLC fractions (F1-F9) were analyzed by RP-HPLC using a Waters BEH300 C4 column (1.7 μm particle size, 2.1 mm × 50 mm) and eluted with a 0.1% TFA-containing mobile phase and a gradient of 1-propanol at 75°C with detection at 215 nm. IgG2-A, IgG2-A / B, and IgG2-B correspond to the various disulfide isoforms of Erenumab. The structures of the various disulfide isoforms are shown schematically in Figures 15A-15C. [Figure 14]Figure 1 shows the Lys-C peptide maps of non-reduced (top trace) and reduced (bottom trace) Aimovig. Aimovig bulk drug was denatured and digested with endoprotease Lys-C. The labeled peaks in the reduced map identify peptides that are present in the reduced Lys-C peptide map but not in the non-reduced Lys-C peptide map. A description of each labeled peptide is listed in Tables 18 and 19. [Figure 15A] FIG. 1 is a schematic diagram of the erenumab disulfide isoform IgG2-A structure identified by non-reduced and reduced Lys-C peptide maps. [Figure 15B] FIG. 1 is a schematic diagram of erenumab disulfide isoform IgG2-A / B structure identified by non-reduced and reduced Lys-C peptide maps. [Figure 15C] FIG. 1 is a schematic diagram of the erenumab disulfide isoform IgG2-B structure identified by non-reduced and reduced Lys-C peptide maps. [Figure 16A] 15A-15C show non-reduced RP-HPLC profiles of erenumab drug substance on an expanded scale. Erenumab drug substance was analyzed by RP-HPLC using a Waters BEH300 C4 column (1.7 μm particle size, 2.1 mm × 50 mm) and eluted with a mobile phase containing 0.1% TFA and a gradient of 1-propanol at 75°C with detection at 215 nm. IgG2-A, IgG2-A / B, and IgG2-B correspond to the various disulfide isoforms of erenumab. The structures of the various disulfide isoforms are shown schematically in Figures 15A-15C. [Figure 16B] Figure 1 shows an overlay of the RP-HPLC profiles of disulfide isoform variant-enriched CEX-HPLC fractions for erenumab drug substance. Erenumab drug substance and CEX-HPLC fractions were analyzed by RP-HPLC using a Waters BEH300 C4 column (1.7 μm particle size, 2.1 mm × 50 mm) and eluted with a mobile phase containing 0.1% TFA and a gradient of 1-propanol at 75°C with detection at 215 nm. [Figures 17A-17B]Representative SE-UHPLC profiles of erenumab drug substance at full scale (FIG. 17A) and expanded scale (FIG. 17B) are shown. Erenumab drug substance was analyzed by SE-UHPLC using a 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 mobile phase and detection at 280 nm absorbance. The buffer peak is indicated by an asterisk (*). [Figure 18]

[0033] Figure 1 shows an overlay of SE-UHPLC collected fractions and erenumab drug substance analyzed by SE-UHPLC. Erenumab drug substance and the fractions collected from semi-preparative SE-HPLC were analyzed by SE-UHPLC using a 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 mobile phase and detection at 280 nm absorbance. Each panel is a plot of absorbance at 280 nm versus elution time in minutes for each fraction overlaid on the SE-UHPLC profile for erenumab drug substance. An overlay of all three SE-UHPLC fractions with erenumab drug substance is shown in panel 4. [Figure 19] rCE-SDS profiles of SE-UHPLC collected fractions and erenumab drug substance at an expanded scale. Erenumab drug substance (DS) and SE-UHPLC fractions (main, HMW1, HMW2) were denatured and reduced before electrokinetic injection into a bare fused silica capillary filled with SDS gel buffer at 25°C. Absorbance was monitored at 220 nm. System peaks are indicated by asterisks (*). LMW = low molecular weight species; MMW = medium molecular weight species; HMW = high molecular weight species; LC = light chain; HC = heavy chain; NGHC = non-glycosylated heavy chain; post-HC = post-heavy chain. [Figure 20] 1 shows the SE-UHPLC profile of erenumab drug substance exposed to heat stress. Erenumab drug substance incubated at 50°C for up to 14 days was analyzed by SE-UHPLC using a 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 mobile phase and detection at 280 nm absorbance. The top trace corresponds to the 14-day time point. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to the identification and characterization of erenumab variants that exhibit reduced functional activity. In particular, erenumab variants with certain structural modifications are much less potent than erenumab in blocking the activation of CGRP receptors by CGRP ligands. Such variants, each of which is described in more detail herein, include isomerized variants, deamidated variants, disulfide isoform variants, acidic variants, and HMW species. By limiting the amount of these variants in erenumab drug substance and erenumab drug products, the overall efficacy of the erenumab composition can be maintained or can be comparable to erenumab compositions utilized in clinical trials and observed to have clinical efficacy. Thus, the present invention provides compositions comprising erenumab and one or more erenumab variants, wherein the amount of erenumab variants in the composition is controlled within a specific range or limit as described herein.

[0018] The term erenumab refers to an IgG2 antibody comprising a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 6. In one embodiment, erenumab comprises a heavy chain comprising the sequence of SEQ ID NO: 1 and a light chain comprising the sequence of SEQ ID NO: 2. In such an embodiment, erenumab is an antibody comprising two heavy chains and two light chains, wherein each heavy chain comprises the sequence of SEQ ID NO: 1 and each light chain comprises the sequence of SEQ ID NO: 2. When produced recombinantly, erenumab may undergo common post-translational modifications at the ends of the heavy and light chains, such as removal of the C-terminal lysine residue at position 456 of the heavy chain and cyclization of the N-terminal glutamine residue in the light and heavy chains to pyroglutamic acid. Thus, the term erenumab may refer to an IgG2 antibody lacking a C-terminal lysine residue in one or both heavy chains and / or comprising a pyroglutamic acid residue as the N-terminal residue instead of a glutamine residue in one or both light chains and / or one or both heavy chains. For example, in some embodiments, erenumab is an antibody comprising two heavy chains and two light chains, wherein each of the heavy chains comprises the sequence of SEQ ID NO: 3 and each of the light chains comprises the sequence of SEQ ID NO: 4. In other embodiments, erenumab is an antibody comprising two heavy chains and two light chains, wherein each of the heavy chains comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and each of the light chains comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0019] In certain embodiments, the erenumab variants present in the compositions of the present invention are charge variants. Charge variants refer to erenumab variants with different charge profiles resulting from post-translational modifications that directly alter the net charge of the antibody, induce conformational changes, or affect local charge distribution. Such post-translational modifications may include deamidation, isomerization, glycation, oxidation, and sialic acid glycosylation. Charge variants can be separated from erenumab and detected using ion exchange chromatography, reversed-phase chromatography, or hydrophobic interaction chromatography. Charge variants of erenumab include, but are not limited to, isomerization variants, deamidation variants, disulfide isoform variants, acidic variants, and glycosylation variants.

[0020] In some embodiments, the charge variant of Aimovig is an isomerization variant. An isomerization variant of Aimovig refers to a variant of Aimovig in which one or more aspartic acid residues in the heavy or light chain polypeptide are converted to isoaspartic acid or succinimide. In one embodiment, an isomerization variant of Aimovig has an isoaspartic acid residue or succinimide at amino acid position 105 in one or both heavy chains of Aimovig (SEQ ID NO: 1 or SEQ ID NO: 3). As described in Example 1, converting the aspartic acid residue at position 105 in CDR3 of the heavy chain variable region to isoaspartic acid significantly reduces the inhibitory potency of Aimovig.

[0021] In other embodiments, the charge variant of erenumab is a desamido variant. A desamido variant of erenumab refers to a variant of erenumab in which one or more asparagine residues in the heavy or light chain polypeptides are converted to aspartic acid and then converted to succinimide or isoaspartic acid. In one embodiment, the desamido variant has the asparagine residue at amino acid position 102 in one or both heavy chains of erenumab (SEQ ID NO: 1 or SEQ ID NO: 3) converted to aspartic acid, succinimide, or isoaspartic acid. As described in Example 1, deamidation of the asparagine residue at position 102 in CDR3 of the heavy chain variable region significantly reduces the inhibitory potency of erenumab. In another embodiment, the desamido variant has the asparagine residue at amino acid positions 393 and / or 398 in one or both heavy chains of erenumab converted to aspartic acid, succinimide, or isoaspartic acid.

[0022] Methods for detecting and quantifying isomerization variants and deamidation variants of erenumab include hydrophobic interaction chromatography, such as the hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) method described in Example 1, peptide mapping, such as the ESI-MS / MS peptide mapping method described in Example 1, ion exchange chromatography, such as the cation exchange-high performance liquid chromatography (CEX-HPLC) method described in Example 3, reversed-phase high performance liquid chromatography (RP-HPLC), and other methods known to those skilled in the art, for example, Kameoka et al., Journal of Biochemistry, Vol. 134:129-135, 2003; Zhang et al., Journal of Pharmaceutical and Biomedical Analysis, Vol. 30:1479-1490, 2003; Yang et al., Electrophoresis, Vol. 31:1764-1772, 2010; Faseri et al., Journal of Chromatography A, Vol. 1498:215-223, 2017; and Leblanc et al., Journal of Chromatography B, Vol. 1048:130-139, 2017.

[0023] The compositions of the present invention contain controlled amounts of isomerized and desamido variants of erenumab such that the overall efficacy of the erenumab composition is maintained at a level observed to have clinical efficacy. See Examples 1 and 2. For example, in certain embodiments, the present invention provides a composition comprising erenumab and one or more erenumab variants, wherein the one or more erenumab variants include isomerized and desamido variants, and the amount of isomerized and desamido variants in the composition is less than about 30%. The amount of isomerized and desamido variants in the composition can be less than about 25%, less than about 20%, less than about 17%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, or less than about 4%. In one embodiment, the amount of isomerized and desamido variants in the composition is less than about 15%. In another embodiment, the amount of isomerized and desamido variants in the composition is less than about 8%. In yet another embodiment, the amount of isomerized variants and deamidated variants in the composition is less than about 4%. In some embodiments, the amount of isomerized variants and deamidated variants in the composition can be about 0.5% to about 30%, about 1% to about 20%, about 1% to about 17%, about 1% to about 15%, about 1% to about 12%, about 1% to about 10%, about 0.5% to about 8%, about 0.5% to about 6%, about 1% to about 4%, about 0.5% to about 3.5%, about 1.5% to about 2.5%, or about 1.7% to about 2.1%. In one embodiment, the amount of isomerized variants and deamidated variants in the composition is about 1% to about 10%. In another embodiment, the amount of isomerized variants and deamidated variants in the composition is about 1% to about 4%. In yet another embodiment, the amount of isomerized and deamidated variants in the composition is from about 0.5% to about 3.5%.

[0024] The amount of isomerized variants and deamidated variants of erenumab in the compositions of the present invention can be determined by any of the above-mentioned methods for detecting and quantifying these variants. In certain embodiments, the amount of isomerized variants and deamidated variants in the composition is determined by HIC-HPLC. Isomerized variants (e.g., isomerization of Asp105 in the heavy chain) and deamidated variants (e.g., deamidation of Asn102 in the heavy chain) are the predominant species in the pre-peak fraction of HIC-HPLC. See Examples 1 and 2. Therefore, the amount of these variants in an erenumab composition can be determined from the percentage of the peak area of ​​the pre-peak in an HIC-HPLC chromatogram. The pre-peak in an HIC-HPLC chromatogram refers to a peak with a height above the detection limit, which usually has a retention time shorter than the retention time of the main peak, which is the peak in the chromatogram with the greatest peak height. See Figures 2A, 2B, 5, 7, and 8. The main peak corresponds to erenumab (e.g., an antibody comprising two heavy chains having the sequences of SEQ ID NO: 1 or 3 and two light chains having the sequences of SEQ ID NO: 2 or 4), which is the predominant form of the antibody molecule in the composition. The percentage of the peak area of ​​the pre-peak can be calculated according to the following formula:

number

number

[0025] In certain embodiments, the amount of isomerization variants (e.g., isomerization of Asp105 in the heavy chain) and deamidation variants (e.g., deamidation of Asn102 in the heavy chain) in the compositions of the invention is about 15% or less, as measured by pre-peak in HIC-HPLC. In one embodiment, the amount of isomerization variants and deamidation variants in the composition is about 10% or less, as measured by pre-peak in HIC-HPLC. In another embodiment, the amount of isomerization variants and deamidation variants in the composition is about 8% or less, as measured by pre-peak in HIC-HPLC. In another embodiment, the amount of isomerization variants and deamidation variants in the composition is about 6% or less, as measured by pre-peak in HIC-HPLC. In yet another embodiment, the amount of isomerization variants and deamidation variants in the composition is about 4% or less, as measured by pre-peak in HIC-HPLC. In yet another embodiment, the amount of isomerized variants and desamido variants in the composition is about 3.2% or less, as measured by pre-peak in HIC-HPLC. In another embodiment, the amount of isomerized variants and desamido variants in the composition is about 2.7% or less, as measured by pre-peak in HIC-HPLC. In some embodiments, the amount of isomerized variants and desamido variants in the composition is about 1% to about 10%, as measured by pre-peak in HIC-HPLC. In other embodiments, the amount of isomerized variants and desamido variants in the composition is about 1% to about 4%, as measured by pre-peak in HIC-HPLC. In still other embodiments, the amount of isomerized variants and desamido variants in the composition is about 0.5% to about 3.5%, as measured by pre-peak in HIC-HPLC.

[0026] In some embodiments, the compositions of the invention comprise erenumab and one or more erenumab variants, including isomerized and desamidated variants; and (i) the amount of Aimovig in the composition is about 90% or greater, about 92% or greater, about 93.9% or greater, about 94.4% or greater, about 96.2% or greater, about 96.8% or greater, or about 97.3% or greater, as measured by main peak in HIC-HPLC; and (ii) the amount of isomerized and deamidated variants in the composition is about 10% or less, about 8% or less, about 6.1% or less, about 5.6% or less, about 3.8% or less, about 3.2% or less, or about 2.7% or less, as measured by pre-peak in HIC-HPLC.

[0027] In one embodiment, the amount of Aimovig in the composition is about 92% or more as measured by the main peak in HIC-HPLC, and the amount of isomerized variants and desamido variants in the composition is about 8% or less as measured by the pre-peak in HIC-HPLC. In another embodiment, the amount of Aimovig in the composition is about 96.8% or more as measured by the main peak in HIC-HPLC, and the amount of isomerized variants and desamido variants in the composition is about 3.2% or less as measured by the pre-peak in HIC-HPLC. In yet another embodiment, the amount of Aimovig in the composition is about 97.3% or more as measured by the main peak in HIC-HPLC, and the amount of isomerized variants and desamido variants in the composition is about 2.7% or less as measured by the pre-peak in HIC-HPLC. In these and other embodiments, HIC-HPLC is performed as described in Example 1, and the amount is determined from the percentage of the peak area for the main peak and pre-peak.

[0028] As described in Example 2, isomerization of Asp105 in heavy chain CDR3 is highly sensitive to forced heat stress conditions, and deamidation in both heavy chain CDR3 (Asn102) and the Fc region (Asn393 and Asn398) increased upon exposure to high pH (e.g., pH 7.4 or higher). Therefore, the production of isomerized and deamidated variants of Aimovig can be controlled during the manufacturing process and storage by limiting the exposure time of Aimovig compositions to temperatures above room temperature and basic solutions, e.g., solutions having a pH above 7.0. For example, in some embodiments, pool holding during manufacturing or storage of Aimovig compositions is kept at room temperature for 14 days or less. In other embodiments, long-term storage of Aimovig compositions is carried out at temperatures below 15°C, e.g., between 2°C and 8°C. Isomerized and desamido variants of erenumab can also be removed from erenumab compositions through purification processes such as hydrophobic interaction chromatography or ion exchange chromatography. Both isomerized and desamido variants of erenumab elute earlier than erenumab in HIC, and therefore these variants can be removed from erenumab compositions by collecting and discarding these early-eluting fractions. Desamido variants of erenumab generally have more negative charges than erenumab, and therefore will elute earlier than erenumab in cation exchange chromatography or later than erenumab in anion exchange chromatography. Therefore, the more acidic desamido variants can be removed from erenumab compositions by appropriately adjusting the collection time of fractions eluting from the cation exchange or anion exchange resin.

[0029] In certain embodiments, the charge variant of erenumab in the composition of the present invention is an acidic variant. An acidic variant refers to a variant of erenumab that has gained a negative charge or lost a positive charge, or has a change in surface charge profile due to a conformational change, thereby having a more acidic nature compared to erenumab. The acidic variant of erenumab exhibits reduced inhibitory potency compared to erenumab. See Example 3 and Table 14. The acidic variant can be separated, detected, and quantified from erenumab using any method that separates proteins based on charge characteristics, such as isoelectric focusing gel electrophoresis, capillary isoelectric focusing gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX). When analyzed by ion exchange chromatography, the acidic variant can be identified by its retention time relative to the main peak corresponding to erenumab. For example, the acidic variant elutes earlier than the main peak from CEX, i.e., the acidic variant has a retention time shorter than that of the main peak in CEX. When using AEX, the acidic variant elutes later than the main peak and therefore has a retention time longer than that for the main peak in AEX.

[0030] In some embodiments, the present invention provides compositions comprising a controlled amount of acidic variants of erenumab, such that the overall efficacy of the erenumab composition is maintained at a level observed to have clinical efficacy. See Example 3. Accordingly, in certain embodiments, the present invention provides compositions comprising erenumab and one or more acidic variants of erenumab, wherein the amount of acidic variants in the composition is less than about 40%. The amount of acidic variants in the composition can be less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, or less than about 26%. In one embodiment, the amount of acidic variants in the composition is less than about 38%. In another embodiment, the amount of acidic variants in the composition is less than about 35%. In another embodiment, the amount of acidic variants in the composition is less than about 32%. In yet another embodiment, the amount of acidic variant in the composition is less than about 30%. In some embodiments, the amount of acidic variant in the composition can be about 20% to about 40%, about 25% to about 38%, about 24% to about 35%, about 28.5% to about 37.5%, about 26% to about 34%, about 28% to about 32%, about 32.5% to about 37.5%, about 28.7% to about 31.3%, or about 26.5% to about 33.6%. In one embodiment, the amount of acidic variant in the composition is about 25% to about 38%. In another embodiment, the amount of acidic variant in the composition is about 26% to about 34%. In yet another embodiment, the amount of acidic variant in the composition is about 28% to about 32%.

[0031] The amount of acidic variants of erenumab in the compositions of the present invention can be determined by any of the above-mentioned methods for detecting and quantifying these variants. In certain embodiments, the amount of acidic variants in the composition is determined by ion exchange chromatography. In one specific embodiment, the amount of acidic variants in the composition is determined by CEX-HPLC, such as the method described in Example 3. When separated by CEX, the acidic variants of erenumab elute earlier than erenumab (the main peak), and the basic variants of erenumab elute later than erenumab (the main peak). See Figure 11. The amount of acidic variants in erenumab compositions can be determined from the percentage of the peak area of ​​the acidic peak in the CEX-HPLC chromatogram. Acidic peaks are those peaks with a retention time shorter than that of the main peak and a peak height greater than the detection limit. Basic peaks are those peaks with a retention time longer than that of the main peak and a peak height greater than the detection limit. The percentage of the peak area for a desired component (e.g., an acidic peak, a main peak, or a basic peak) can be calculated by dividing the peak area for the desired component (e.g., an acidic peak, a main peak, or a basic peak) by the total integrated peak area and multiplying the result by 100. In certain embodiments, the CEX-HPLC method is performed as described in Example 3.

[0032] In some embodiments, a composition of the invention comprises Aimovig and one or more Aimovig variants, wherein the Aimovig variants include an acidic variant, and the amount of the acidic variant in the composition is about 40% or less, about 38% or less, about 36% or less, about 35% or less, about 34% or less, or about 32% or less, as measured by the acidic peak in CEX-HPLC. In one embodiment, the amount of the acidic variant in the composition is about 25% to about 38%, as measured by the acidic peak in CEX-HPLC. In another embodiment, the amount of the acidic variant in the composition is about 26% to about 34%, as measured by the acidic peak in CEX-HPLC. In yet another embodiment, the amount of the acidic variant in the composition is about 26.5% to about 33.6%, as measured by the acidic peak in CEX-HPLC. In yet another embodiment, the amount of acidic variant in the composition is about 28.7% to about 31.3%, as measured by the acidic peak in CEX-HPLC. In another embodiment, the amount of acidic variant in the composition is about 32.5% to about 37.5%, as measured by the acidic peak in CEX-HPLC. In these and other embodiments, CEX-HPLC is performed as described in Example 3, and the amount of acidic variant is determined from the percentage of the peak area for the acidic peak.

[0033] Acidic variants may include disulfide isoform variants, deamidated variants, fragmented variants, non-consensus glycosylation variants, high molecular weight (HMW) species, and combinations thereof. In certain embodiments, acidic variants include disulfide isoform variants (described in more detail herein), fragmented variants, or combinations thereof. As described in Example 3, acidic fractions enriched in fragmented variants or disulfide isoform variants, particularly the IgG2-B disulfide isoform, exhibit reduced potency compared to erenumab.

[0034] In some embodiments, acidic variants include fragmented variants. Fragmented variants can be formed by hydrolysis of peptide bonds, resulting in cleavage of the light and / or heavy chain polypeptides. Thus, fragmented variants of erenumab refer to fragments of the erenumab light chain (SEQ ID NO: 2 or 4) or the erenumab heavy chain (SEQ ID NO: 1 or 3) that consist of fewer amino acids than the full-length chain. Fragmented variants can be measured using reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS), such as the rCE-SDS method described in Example 3, which separates these fragmented variants into low molecular weight (LMW) and medium molecular weight (MMW) species. The LMW species of erenumab are fragments with a molecular weight of approximately 22,790 Da, which is less than that of the intact erenumab light chain. The MMW species of erenumab is a fragment with a molecular weight of about 50,165 Da (deglycosylated), which is larger than that of the intact erenumab light chain but less than that of the intact erenumab heavy chain.When separated by rCE-SDS, the LMW species of erenumab migrates before the erenumab light chain peak, and the MMW species of erenumab migrates between the erenumab light chain peak and the erenumab heavy chain peak.See, for example, Figure 19.

[0035] The amount of acidic variants in erenumab composition can be reduced by using chromatographic methods that separate proteins based on charge characteristics, such as ion exchange chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography.For example, refer to the methods described in U.S. Patent Nos. 8,946,395; 9,067,990; 9,249,218; and 9,346,879.As mentioned above, when separated by cation exchange chromatography, acidic variants of erenumab elute earlier than erenumab (main peak).Therefore, acidic variants can be collected and discarded before the elution of erenumab main peak, or the collection of erenumab fraction can be started after the elution of acidic variants. When separated by anion exchange chromatography, the acidic variant of Aimovig elutes later than Aimovig (the main peak), and therefore the acidic variant can be reduced or removed from the Aimovig composition by stopping the collection of the Aimovig fraction before the elution of the acidic variant.

[0036] In certain embodiments, acidic variants include disulfide isoform variants. IgG2 antibodies can exhibit different structural isoforms based on the arrangement of disulfide bonds in the hinge region. In the classical disulfide isoform known as IgG2-A, the Fab arms are not linked to the hinge region via disulfide bonds, and four cysteine ​​residues in the hinge region of one heavy chain (e.g., C232, C233, C236, and C239 in SEQ ID NO: 1 or 3) each form a disulfide bond with the corresponding cysteine ​​residue in the hinge region of the other heavy chain. See Figure 15A. Disulfide isoform variants refer to variants of IgG2 antibodies that have disulfide bond connectivity that differs from that found in the classical IgG2-A isoform. In some embodiments, the disulfide isoform variant is the IgG2-B isoform. In the IgG2-B isoform structure, both Fab arms are linked to the hinge region by one or more cysteine ​​residues in the hinge region that form disulfide bonds with cysteine ​​residues in the heavy chain constant CH1 region and / or the light chain constant CL region. See Figure 15C. For example, in one embodiment, the IgG2-B isoform comprises the following disulfide bond connectivity between a first heavy chain (HC1), a first light chain (LC1), a second heavy chain (HC2), and a second light chain (LC2) having amino acid positions relative to SEQ ID NO: 1 or 3 for the heavy chain and SEQ ID NO: 2 or 4 for the light chain: C232 of HC1 versus C157 of HC2 C233 of HC1 versus C215 of LC1 C232 in HC2 versus C157 in HC1 C233 for HC2 versus C215 for LC2 C236 of HC1 vs. C236 of HC2 C239 in HC1 vs. C239 in HC2

[0037] In another embodiment, the disulfide isoform variant is an IgG2-A / B isoform. In the IgG2-A / B isoform structure, only one Fab arm is linked to the hinge region by one or more cysteine ​​residues in the hinge region that form disulfide bonds with cysteine ​​residues in the heavy chain constant CH1 region and / or the light chain constant CL region. See Figure 15B. In one embodiment, the IgG2-A / B isoform comprises the following disulfide bond connectivity between HC1, LC1, HC2, and LC2 having amino acid positions relative to SEQ ID NO: 1 or 3 for the heavy chain and SEQ ID NO: 2 or 4 for the light chain: C232 of HC1 versus C157 of HC2 C232 for HC2 versus C215 for LC2 C233 in HC1 vs. C233 in HC2 C236 of HC1 vs. C236 of HC2 C239 in HC1 vs. C239 in HC2

[0038] The present invention includes compositions comprising Aimovig and one or more disulfide isoform variants thereof. The disulfide isoform variants may include the IgG2-B isoform, the IgG2-A / B isoform, or a combination of the two. In some embodiments, a composition comprises Aimovig and one or more disulfide isoform variants thereof, wherein the one or more disulfide isoform variants include the IgG2-B isoform, and the amount of IgG2-B isoform in the composition is less than about 20%. The amount of IgG2-B isoform in the composition may be less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less than about 6%. In one embodiment, the amount of IgG2-B isoform in the composition is less than about 10%. In another embodiment, the amount of IgG2-B isoform in the composition is less than about 8%. In certain embodiments, the amount of IgG2-B isoform in the composition can be about 0.5% to about 20%, about 1% to about 16%, about 3% to about 12%, about 0.5% to about 8%, about 1% to about 6%, about 4% to about 6%, about 4.4% to about 5.9%, or about 4.6% to about 5.2%. In one embodiment, the amount of IgG2-B isoform in the composition is about 0.5% to about 8%. In another embodiment, the amount of IgG2-B isoform in the composition is about 4% to about 6%.

[0039] In certain embodiments, the compositions of the present invention comprise erenumab and one or more disulfide isoform variants thereof, wherein the one or more disulfide isoform variants comprise the IgG2-A / B isoform. The amount of the IgG2-B isoform in the composition can be about 10% to about 56%, about 20% to about 40%, about 26% to about 38%, about 27% to about 32%, about 32% to about 38%, about 34% to about 37%, or about 34.2% to about 35.5%. In one embodiment, the amount of the IgG2-A / B isoform in the composition is about 32% to about 38%. In another embodiment, the amount of the IgG2-A / B isoform in the composition is about 34% to about 37%.

[0040] In some embodiments, the compositions of the invention comprise Aimovig IgG2-A isoform, Aimovig IgG2-A / B isoform, and Aimovig IgG2-B isoform, (i) the amount of IgG2-A isoform in the composition is between about 50% and about 70%, between about 52% and about 66%, between about 56% and about 60%, or between about 57.4% and about 59.3%; (ii) the amount of IgG2-A / B isoform in the composition is between about 26% and about 38%, between about 32% and about 38%, between about 34% and about 37%, or between about 34.2% and 35.5%; and (iii) The amount of IgG2-B isoform in the composition is about 3% to about 12%, about 1% to about 10%, about 4% to about 8%, or about 4.6% to about 5.2%.

[0041] Methods for detecting and quantifying disulfide isoforms include non-reducing reversed-phase high performance liquid chromatography (RP-HPLC), such as the RP-HPLC method described in Examples 3 and 4 and in Wypych et al., Journal of Biological Chemistry, Vol. 283(23):16194-16205, 2008; peptide mapping, such as the Lys-C peptide mapping method described in Example 4; and mass spectrometry-based methods, such as those described in Zhang et al., Anal Chem., Vol. 82(3):1090-1099, 2010 and Zhang et al., Biochemistry, Vol. 54:1956-1962, 2015. In certain embodiments, the amount of disulfide isoforms (e.g., IgG2-A, IgG2-A / B, and IgG2-B) in a composition of the invention is determined by non-reducing RP-HPLC, optionally using the peak area percentage for each of the isoforms in the RP-HPLC profile (see, e.g., Figure 16A).

[0042] The ratio of disulfide isoforms can be altered by exposing a composition containing different isoforms to a reducing-oxidizing (redox) agent alone or in combination with a chaotropic agent using the methods described in Dillon et al., Journal of Biological Chemistry, Vol. 283(23):16206-16215, 2008, WO 2006 / 047340, or WO 2006 / 060083, which are hereby incorporated by reference in their entireties. For example, the ratio of the IgG2-A isoform can be increased by exposing a composition containing different isoforms to a redox agent (e.g., cysteine / cystine or glutathione / oxidized glutathione) and a chaotropic agent (e.g., guanidine hydrochloride). Thus, in some embodiments, the amount of erenumab's IgG2-B isoform, which is significantly less potent than the IgG2-A / B and IgG2-A isoforms, can be optionally reduced or removed from the composition by exposing the composition to an oxidation-reducing agent in combination with a chaotropic agent, according to the methods described in Dillon et al., 2008; WO 2006 / 047340; or WO 2006 / 060083.

[0043] In certain embodiments, the erenumab variants present in the compositions of the present invention are size variants. A size variant of erenumab refers to a variant having either a lower molecular weight than the erenumab monomer or a higher molecular weight than the erenumab monomer. As used herein, the term erenumab monomer refers to an intact antibody comprising two heavy chains and two light chains, with the monomer having a molecular weight of approximately 146,194 Da when measuring the deglycosylated heavy and light chains without N- or C-terminal modifications. Size variants include the above-mentioned LMW and MMW species, as well as fragmented variants such as high molecular weight (HMW) species. The HMW species of erenumab refers to species having a molecular weight greater than the erenumab monomer. HMW species may include dimers, multimers, and other aggregate forms of erenumab formed by covalent and non-covalent self-association. In some embodiments, the HMW species of Aimovig comprises a covalently linked dimer of Aimovig (i.e., two covalently linked Aimovig monomers). In certain embodiments, the covalently linked Aimovig dimer is a reducible dimer, where the dimer is reduced to its individual heavy and light chains under reducing and denaturing conditions.

[0044] Methods for detecting and quantifying Aimovig size variants include size exclusion chromatography, such as the size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method described in Example 5, capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) performed under reducing or non-reducing conditions, such as the rCE-SDS and nrCE-SDS methods described in Example 3, sedimentation velocity ultracentrifugation, and SE-UHPLC with static light scattering detection to determine molar mass.

[0045] Fractions enriched in HMW species are significantly less potent than erenumab in inhibiting ligand-induced activation of the CGRP receptor. See Example 5 and Table 26. Thus, the present invention provides erenumab compositions containing controlled amounts of HMW species such that the overall efficacy of the erenumab composition is maintained at a level observed to have clinical efficacy. In some embodiments, the compositions of the present invention comprise erenumab and HMW species of erenumab, wherein the amount of HMW species in the composition is less than about 3.0%. The amount of HMW species in the composition can be about 2.5% or less, about 2.4% or less, about 2.3% or less, about 2.2% or less, about 2.1% or less, about 2.0% or less, about 1.8% or less, about 1.6% or less, about 1.4% or less, about 1.2% or less, about 1.0% or less, about 0.8% or less, about 0.6% or less, or about 0.4% or less. In one embodiment, the amount of HMW species in the composition is about 1.8% or less. In another embodiment, the amount of HMW species in the composition is about 1.4% or less. In yet another embodiment, the amount of HMW species in the composition is about 1.2% or less. In yet another embodiment, the amount of HMW species in the composition is about 0.6% or less. In some embodiments, the amount of HMW species in the composition can be about 0.3% to about 2.4%, about 0.4% to about 1.2%, about 0.6% to about 2.1%, about 0.3% to about 1.8%, about 1.0% to about 1.6%, or about 0.6% to about 1.4%. In one embodiment, the amount of HMW species in the composition is about 0.4% to about 1.2%. In another embodiment, the amount of HMW species in the composition is about 0.6% to about 2.1%.

[0046] The amount of size variants in the compositions of the present invention can be determined by any of the above-mentioned methods for detecting and quantifying these variants. In certain embodiments, the amount of size variants (e.g., HMW species) is determined by SE-UHPLC. The HMW species of Aimovig elutes earlier than the Aimovig monomer (main peak) in SE-UHPLC and therefore corresponds to the pre-peak in the SE-UHPLC chromatogram, while the LMW and MMW species elute later than the Aimovig monomer and therefore correspond to the post-peak in the SE-UHPLC chromatogram. See Example 5 and Figures 17A and 17B. Therefore, the amount of these size variants in Aimovig compositions can be determined from the percentage of the peak area of ​​the pre-peak (HMW species) or post-peak (LMW and MMW species) in the SE-UHPLC chromatogram. In some embodiments, the SE-UHPLC method is performed as described in Example 5.

[0047] In certain embodiments, the compositions of the invention comprise Aimovig and one or more Aimovig size variants, wherein the Aimovig size variants comprise a HMW species of Aimovig; and (i) the amount of Aimovig in the composition is about 97.5% or greater, about 97.9% or greater, about 98.2% or greater, about 98.4% or greater, about 98.6% or greater, about 98.8% or greater, about 99.2% or greater, or about 99.4% or greater, as measured by main peak in SE-UHPLC; and (ii) the amount of HMW species in the composition is about 2.5% or less, about 2.1% or less, about 1.8% or less, about 1.6% or less, about 1.4% or less, about 1.2% or less, about 0.8% or less, or about 0.6% or less, as measured by pre-peak in SE-UHPLC.

[0048] In one embodiment, the amount of Aimovig in the composition is about 97.9% or more, as measured by the main peak in SE-UHPLC, and the amount of HMW species in the composition is about 2.1% or less, as measured by the pre-peak in SE-UHPLC. In another embodiment, the amount of Aimovig in the composition is about 98.2% or more, as measured by the main peak in SE-UHPLC, and the amount of HMW species in the composition is about 1.8% or less, as measured by the pre-peak in SE-UHPLC. In yet another embodiment, the amount of Aimovig in the composition is about 98.8% or more, as measured by the main peak in SE-UHPLC, and the amount of HMW species in the composition is about 1.2% or less, as measured by the pre-peak in SE-UHPLC. In yet another embodiment, the amount of Aimovig in the composition is about 98.6% or greater, as measured by the main peak in SE-UHPLC, and the amount of HMW species in the composition is about 1.4% or less, as measured by the pre-peak in SE-UHPLC. In these and other embodiments, SE-UHPLC is performed as described in Example 5, and the amount is determined from the peak area percentages for the main peak and pre-peak.

[0049] The amount of size variants (e.g., HMW species) in erenumab compositions can be reduced using methods that separate proteins based on size or hydrodynamic volume, such as size exclusion chromatography and filtration. In addition, affinity chromatography (e.g., protein A chromatography), cation exchange chromatography, hydrophobic interaction chromatography, and mixed-format chromatography can also effectively reduce HMW species. See, for example, Shukla et al., Journal of Chromatography B, Vol. 848: 28-39, 2007; Chen et al., Journal of Chromatography A, Vol. 1217: 216-224, 2010; U.S. Patent Nos. 6,620,918; 9,505,803; and 9,783,570. As described above, the HMW species of erenumab elute earlier than erenumab (main peak) when separated by size exclusion chromatography. Thus, the HMW species can be collected and discarded before the elution of the erenumab main peak, or collection of the erenumab fraction can begin after the elution of the HMW species.

[0050] The erenumab compositions of the present invention can be analyzed for one or more of the erenumab variants described herein. For example, in some embodiments, the present invention includes compositions comprising erenumab and one or more erenumab variants, where the erenumab variants include isomerized variants, desamidated variants, acidic variants, HMW species, or combinations thereof, and the compositions have controlled amounts of each of these variants. In one embodiment, the composition comprises Aimovig and one or more Aimovig variants, wherein the Aimovig variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof, and the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 25% to about 38% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 2.1% or less as measured by SE-UHPLC; and (c) the amount of isomerized variants and desamidated variants in the composition is about 8.0% or less as measured by pre-peak in HIC-HPLC. In another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is less than about 38% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 2.5% or less as measured by SE-UHPLC; and (c) the amount of isomerized variants and deamidated variants in the composition is about 10% or less as measured by pre-peak in HIC-HPLC. In another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 32.5% to about 37.5% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 1.8% or less as measured by SE-UHPLC; and (c) the amount of isomerized variants and deamidated variants in the composition is about 6.1% or less as measured by pre-peak in HIC-HPLC.In yet another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 26.5% to about 33.6% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 1.2% or less as measured by SE-UHPLC; and (c) the amount of isomerized variants and deamidated variants in the composition is about 3.2% or less as measured by pre-peak in HIC-HPLC. In yet another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 26.5% to about 33.6% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 1.2% or less as measured by SE-UHPLC; and (c) the amount of isomerized variants and deamidated variants in the composition is about 2.7% or less as measured by pre-peak in HIC-HPLC. In another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 26.5% to about 33.6% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 1.4% or less as measured by SE-UHPLC; and (c) the amount of isomerized and deamidated variants in the composition is about 3.2% or less as measured by pre-peak in HIC-HPLC. In another embodiment, the composition has one or more or all of the following characteristics: (a) the amount of acidic variants in the composition is about 28.7% to about 31.3% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 0.6% or less as measured by SE-UHPLC; and (c) the amount of isomerized and deamidated variants in the composition is about 2.1% or less as measured by pre-peak in HIC-HPLC.

[0051] The erenumab compositions of the invention can be made by recombinantly expressing nucleic acids encoding the heavy and light chains in host cells, partially purifying or purifying erenumab from the host cell culture or host cell lysate, and analyzing the resulting composition for one or more of the erenumab variants detailed herein according to the methods described in detail below.

[0052] For recombinant production of Aimovig, one or more nucleic acids encoding a heavy chain (e.g., a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 1) and a light chain (e.g., a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 2) are inserted into one or more expression vectors. The nucleic acids encoding the heavy chain and the light chain can be inserted into a single expression vector, or they can be inserted into separate expression vectors. As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a particular host cell. Expression vectors may contain sequences that affect or control transcription, translation, and, if present, introns, affect RNA splicing of the operably linked coding region. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence operably linked to the coding sequence of interest can also be encoded by the expression vector, thereby enabling the recombinant host cell to secrete the expressed polypeptide, making it easier to isolate the polypeptide from the cell if desired. The vector can also contain one or more selectable marker genes to facilitate selection of host cells into which the vector has been introduced. Exemplary nucleic acids encoding the heavy and light chains of Aimovig and suitable signal peptide sequences and other components for expression vectors for recombinantly expressing Aimovig are described in WO 2010 / 075238, which is hereby incorporated by reference in its entirety.

[0053] After the expression vector is constructed and one or more nucleic acid molecules encoding the heavy and light chain components of Aimovig are inserted into the appropriate sites of the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the Aimovig expression vector into the selected host cell can be carried out by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell used. These and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook, Fritsch, and Maniatis (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), and Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989).

[0054] When cultured under appropriate conditions, the host cells synthesize Aimovig, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications desired or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0055] Exemplary host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens. Examples of lower eukaryotic host microorganisms include Bacillus species, such as B. marcescens and Shigella, and Bacillus species, such as B. subtilis, B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts suitable for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, species of Pichia, such as P. pastoris, Schizosaccharomyces pombe, and P. cerevisiae, are also suitable. Several other genera, species, and strains are commonly available and useful herein, such as A. pombe, Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger.

[0056] Host cells for the expression of glycosylated antibodies can be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection of such cells, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available.

[0057] Vertebrate host cells are also suitable hosts, and recombinant production of antibodies from such cells is routine. Mammalian cell lines available as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture (Graham et al., J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC mammalian myeloma cells and many other cell lines. CHO cells are preferred host cells in some embodiments for expressing Aimovig.

[0058] To produce Aimovig, host cells are transformed or transfected with the above-described expression vector and cultured in conventional nutrient media modified appropriately for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. The host cells used to produce Aimovig can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; or WO 87 / 00195 may be used as a culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamicin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0059] When the host cells are cultured, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the host cells are lysed (e.g., by mechanical shearing, osmotic shock, or enzymatic methods), and particulate debris (e.g., host cells and lysed fragments) is removed, for example, by centrifugation, microfiltration, or ultrafiltration. If the antibody is secreted into the culture medium, it may be separated from the host cells by centrifugation or microfiltration, and then optionally concentrated by ultrafiltration. Erenumab may be further purified or partially purified using one or more chromatography steps, such as affinity chromatography (e.g., protein A or protein G affinity chromatography), cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography.

[0060] Once an erenumab composition has been purified or obtained, the composition can be evaluated for the presence and quantity of one or more erenumab variants described herein, including isomerized variants, desamidated variants, acidic variants, and size variants (e.g., HMW species). Accordingly, the present invention includes methods for assessing the quality of an erenumab composition, comprising obtaining an erenumab composition containing erenumab and one or more erenumab variants; measuring the amount of one or more erenumab variants in the composition; comparing the measured amount of one or more erenumab variants to a predetermined reference standard; and, if the comparison indicates satisfaction of the predetermined reference standard, producing a pharmaceutical formulation or product of the erenumab composition. In some embodiments, the method includes one, two, or three of: (1) measuring the amount of isomerized variants and desamidated variants in the composition; (2) measuring the amount of acidic variants in the composition; and / or (3) measuring the amount of HMW species in the composition. In certain embodiments, all three measurements are performed on the erenumab composition.

[0061] The predetermined reference standard for each Aimovig variant can be a threshold amount or range of amounts of the variant that does not significantly affect the efficacy of Aimovig compositions for inhibiting ligand-induced activation of the CGRP receptor. For example, the predetermined reference standard for each Aimovig variant can be any of the limits or ranges disclosed herein for each of the variants, because Aimovig compositions having these limits / ranges of variants have efficacy comparable to Aimovig compositions evaluated in clinical trials and shown to have clinical efficacy.

[0062] In certain embodiments of the method, if the measured amount of the erenumab variant in the composition meets a predetermined reference standard, the erenumab composition is classified as acceptable and can proceed to the next step in the manufacturing or distribution process, such as, for example, by making a pharmaceutical formulation of the composition (e.g., by combining with one or more excipients or diluents); by making a pharmaceutical product of the composition (e.g., by filling into a vial, syringe, autoinjector, or other container or delivery device); packaging the composition with instructions for use, diluents, and / or a delivery device; or releasing the composition for marketing or distribution to a distributor. In some embodiments of the method, a pharmaceutical formulation of the erenumab composition is made if the measured amount of the erenumab variant in the composition meets a predetermined reference standard. In other embodiments of the method, a pharmaceutical product of the erenumab composition is made if the measured amount of the erenumab variant in the composition meets a predetermined reference standard. Methods of making pharmaceutical formulations and pharmaceutical products of erenumab compositions are described in more detail below. If the measured amount of erenumab variant in the composition does not meet the predetermined reference standard, in some embodiments of the method, the erenumab composition is classified as unacceptable and may be discarded, destroyed, or subjected to additional manufacturing steps, such as additional purification to remove or reduce the amount of erenumab variant in the composition so that the predetermined reference standard is met.

[0063] In one embodiment, a method for assessing the quality of an erenumab composition includes obtaining an erenumab composition containing erenumab and erenumab isomerization variants and desamidation variants; measuring the amount of the isomerization variants and desamidation variants in the composition; comparing the measured amount of the isomerization variants and desamidation variants with a predetermined reference standard; and producing a pharmaceutical formulation or pharmaceutical product of the erenumab composition if the comparison indicates that the predetermined reference standard is met. The predetermined reference standard for the amount of isomerization variants and desamidation variants in an erenumab composition can be less than about 30%, for example, about 25% or less, about 20% or less, about 17% or less, about 15% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, or about 4% or less. In one embodiment, the predetermined reference standard for the amount of isomerization variants and desamidation variants in an erenumab composition is about 15% or less. In another embodiment, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 10% or less. In another embodiment, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 8% or less. In yet another embodiment, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 6% or less. In yet another embodiment, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 4% or less. In certain embodiments, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 3.2% or less. In other embodiments, the predetermined reference standard for the amount of isomerization variants and desamidation variants in erenumab compositions is about 2.7% or less. In some embodiments, the predetermined reference standard for the amount of isomerization variants and deamidation variants in the erenumab composition can be a range of amounts, for example, about 1% to about 10% of the erenumab composition, about 1% to about 4% of the erenumab composition, or about 0.5% to about 3.5% of the erenumab composition.In certain embodiments, the amount of isomerized and desamidated variants in the erenumab composition is measured by HIC-HPLC, e.g., by the percentage of the peak area of ​​the pre-peak in HIC-HPLC. In such embodiments, the HIC-HPLC method may be performed as described in Example 1.

[0064] In another embodiment, a method for assessing the quality of an erenumab composition includes obtaining an erenumab composition containing erenumab and erenumab acidic variants; measuring the amount of the acidic variants in the composition; comparing the measured amount of the acidic variants with a predetermined reference standard; and producing a pharmaceutical formulation or pharmaceutical product of the erenumab composition if the comparison indicates that the predetermined reference standard is met. The predetermined reference standard for the amount of acidic variants in an erenumab composition can be less than about 40%, for example, about 38% or less, about 37% or less, about 36% or less, about 35% or less, about 34% or less, about 33% or less, about 32% or less, about 31% or less, about 30% or less, about 29% or less, about 28% or less, about 27% or less, or about 26% or less. In one embodiment, the predetermined reference standard for the amount of acidic variants in an erenumab composition is about 38% or less. In another embodiment, the predetermined reference standard for the amount of acidic variants in the erenumab composition is about 35% or less. In yet another embodiment, the predetermined reference standard for the amount of acidic variants in the erenumab composition is about 32% or less. In yet another embodiment, the predetermined reference standard for the amount of acidic variants in the erenumab composition is about 30% or less. In some embodiments, the predetermined reference standard for the amount of acidic variants in the erenumab composition is a range of amounts, such as about 25% to about 38% of the erenumab composition, about 32.5% to about 37.5% of the erenumab composition, about 26% to about 34% of the erenumab composition, or about 26.5% to about 33.6% of the erenumab composition. In one specific embodiment, the predetermined reference standard for the amount of acidic variants in the erenumab composition is about 25% to about 38%. In another specific embodiment, the predetermined reference standard for the amount of acidic variants in the erenumab composition is about 26.5% to about 33.6%. In certain embodiments, the amount of acidic variants in the erenumab composition is measured by CEX-HPLC, for example, by the percentage of the peak area of ​​the acidic peak in CEX-HPLC. In such embodiments, the CEX-HPLC method may be performed as described in Example 3.

[0065] In another embodiment, a method for assessing the quality of an erenumab composition includes obtaining an erenumab composition containing erenumab and HMW species of erenumab; measuring the amount of HMW species in the composition; comparing the measured amount of HMW species to a predetermined reference standard; and producing a pharmaceutical formulation or pharmaceutical product of the erenumab composition if the comparison indicates that the predetermined reference standard is met. The predetermined reference standard for the amount of HMW species in the erenumab composition can be less than about 3.0%, e.g., about 2.5% or less, about 2.4% or less, about 2.3% or less, about 2.2% or less, about 2.1% or less, about 2.0% or less, about 1.8% or less, about 1.6% or less, about 1.4% or less, about 1.2% or less, about 1.0% or less, about 0.8% or less, about 0.6% or less, or about 0.4% or less. In one embodiment, the predetermined reference standard for the amount of HMW species in an erenumab composition is about 2.5% or less. In another embodiment, the predetermined reference standard for the amount of HMW species in an erenumab composition is about 1.8% or less. In another embodiment, the predetermined reference standard for the amount of HMW species in an erenumab composition is about 1.4% or less. In yet another embodiment, the predetermined reference standard for the amount of HMW species in an erenumab composition is about 1.2% or less. In yet another embodiment, the predetermined reference standard for the amount of HMW species in an erenumab composition is about 0.6% or less. The predetermined reference standard for the amount of HMW species in the erenumab composition can, in some embodiments, be a range of amounts, such as about 0.3% to about 2.4% of the erenumab composition, about 0.6% to about 2.1% of the erenumab composition, about 0.4% to about 1.2% of the erenumab composition, or about 0.6% to about 1.4% of the erenumab composition. In certain embodiments, the amount of HMW species in the erenumab composition is measured by SE-UHPLC, for example, by the percentage of the peak area of ​​the pre-peak in the SE-UHPLC. In such embodiments, the SE-UHPLC method can be performed as described in Example 5.

[0066] In certain embodiments of the methods of the present invention, the method comprises: (a) obtaining an erenumab composition comprising erenumab and one or more erenumab variants, wherein the erenumab variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof; (b) the following: (i) measuring the amount of isomerized variants and deamidated variants in the composition by pre-peak in HIC-HPLC, and comparing the measured amount with a predetermined reference standard of about 10% or less; (ii) measuring the amount of the acidic variant in the composition by the acidic peak in CEX-HPLC and comparing the measured amount with a predetermined reference standard of about 25% to about 38%; and / or (iii) evaluating the erenumab composition by performing one, two, or all three of the following: measuring the amount of HMW species in the composition by pre-peak in SE-UHPLC and comparing the measured amount to a predetermined reference standard of about 2.5% or less; and (c) preparing a pharmaceutical formulation or product of the erenumab composition if the comparison in step (b) indicates that the pre-defined reference standard / acceptance standard is met. In some embodiments, all three steps (b)(i), (b)(ii), and (b)(iii) are performed. In other embodiments, only steps (b)(i) and (b)(ii) are performed. In still other embodiments, only steps (b)(ii) and (b)(iii) are performed. In certain embodiments, only steps (b)(i) and (b)(iii) are performed.

[0067] In certain other embodiments of the methods of the present invention, the method comprises: (a) obtaining an erenumab composition comprising erenumab and one or more erenumab variants, wherein the erenumab variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof; (b) the following: (i) measuring the amount of isomerized variants and deamidated variants in the composition by pre-peak in HIC-HPLC, and comparing the measured amount with a predetermined reference standard of about 3.2% or less; (ii) measuring the amount of the acidic variant in the composition by the acidic peak in CEX-HPLC and comparing the measured amount with a predetermined reference standard of about 26.5% to about 33.6%; and / or (iii) evaluating the erenumab composition by performing one, two, or all three of the following: measuring the amount of HMW species in the composition by pre-peak in SE-UHPLC and comparing the measured amount to a predetermined reference standard of about 1.2% or less; and (c) preparing a pharmaceutical formulation or product of the erenumab composition if the comparison in step (b) indicates that the pre-defined reference standard / acceptance standard is met. In some embodiments, all three steps (b)(i), (b)(ii), and (b)(iii) are performed. In other embodiments, only steps (b)(i) and (b)(ii) are performed. In still other embodiments, only steps (b)(ii) and (b)(iii) are performed. In certain embodiments, only steps (b)(i) and (b)(iii) are performed.

[0068] In some embodiments of the methods of the present invention, the method comprises: (a) obtaining an erenumab composition comprising erenumab and one or more erenumab variants, wherein the erenumab variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof; (b) the following: (i) measuring the amount of isomerized variants and deamidated variants in the composition by pre-peak in HIC-HPLC, and comparing the measured amount with a predetermined reference standard of about 2.7% or less; (ii) measuring the amount of the acidic variant in the composition by the acidic peak in CEX-HPLC and comparing the measured amount with a predetermined reference standard of about 26.5% to about 33.6%; and / or (iii) evaluating the erenumab composition by performing one, two, or all three of the following: measuring the amount of HMW species in the composition by pre-peak in SE-UHPLC and comparing the measured amount to a predetermined reference standard of about 1.2% or less; and (c) preparing a pharmaceutical formulation or product of the erenumab composition if the comparison in step (b) indicates that the pre-defined reference standard / acceptance standard is met. In some embodiments, all three steps (b)(i), (b)(ii), and (b)(iii) are performed. In other embodiments, only steps (b)(i) and (b)(ii) are performed. In still other embodiments, only steps (b)(ii) and (b)(iii) are performed. In certain embodiments, only steps (b)(i) and (b)(iii) are performed.

[0069] In another embodiment of the method of the present invention, the method comprises: (a) obtaining an erenumab composition comprising erenumab and one or more erenumab variants, wherein the erenumab variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof; (b) the following: (i) measuring the amount of isomerized variants and deamidated variants in the composition by pre-peak in HIC-HPLC, and comparing the measured amount with a predetermined reference standard of about 3.2% or less; (ii) measuring the amount of the acidic variant in the composition by the acidic peak in CEX-HPLC and comparing the measured amount with a predetermined reference standard of about 26.5% to about 33.6%; and / or (iii) evaluating the erenumab composition by performing one, two, or all three of the following: measuring the amount of HMW species in the composition by pre-peak in SE-UHPLC and comparing the measured amount to a predetermined reference standard of about 1.4% or less; and (c) preparing a pharmaceutical formulation or product of the erenumab composition if the comparison in step (b) indicates that the pre-defined reference standard / acceptance standard is met. In some embodiments, all three steps (b)(i), (b)(ii), and (b)(iii) are performed. In other embodiments, only steps (b)(i) and (b)(ii) are performed. In still other embodiments, only steps (b)(ii) and (b)(iii) are performed. In certain embodiments, only steps (b)(i) and (b)(iii) are performed.

[0070] The methods of the present invention for assessing the quality or evaluating the above-described erenumab compositions can be employed in a variety of contexts. For example, the methods can be used as quality control methods (e.g., as in-manufacturing controls) at various steps in the manufacturing process for erenumab. In some embodiments, the methods can be used upon completion of most or all of the manufacturing process, e.g., as a lot release method for erenumab bulk substance (e.g., active ingredient or API) or erenumab drug product (e.g., API formulated with one or more excipients for human use). The methods can also be used to evaluate erenumab compositions stored for various periods of time to facilitate determining the drug's expiration date. The methods can also be used to reevaluate erenumab compositions that do not initially meet predefined reference standards and have been reprocessed (e.g., subjected to additional purification operations).

[0071] Thus, the erenumab composition employed in the method can be any composition comprising erenumab and, optionally, one or more erenumab variants. In some embodiments, the erenumab composition is obtained from a Chinese hamster ovary (CHO) cell line expressing a nucleic acid encoding a heavy chain of SEQ ID NO: 1 and a nucleic acid encoding a light chain of SEQ ID NO: 2. In such embodiments, the erenumab composition is a cell culture harvest (e.g., a clarified cell culture supernatant or a clarified cell lysate). In other such embodiments, the erenumab composition is a partially purified preparation of erenumab that has been subjected to one or more purification procedures (e.g., a pool or fraction derived from one or more chromatography or filtration steps). In one embodiment, the erenumab composition is an elution pool from a cation exchange chromatography material. In another embodiment, the erenumab composition is erenumab drug substance (e.g., an active ingredient or API). In yet another embodiment, the erenumab composition is an erenumab drug product (e.g., erenumab drug substance or API formulated with one or more excipients for human use).

[0072] The biological activity of an erenumab composition can be evaluated as part of the quality assessment method described above. In some embodiments, the method includes assessing the ability of the erenumab composition to inhibit CGRP ligand-induced activation of the human CGRP receptor. Various assays for assessing CGRP receptor activation are known in the art, including cell-based assays that measure CGRP ligand-induced calcium mobilization and cAMP production. An exemplary cell-based cAMP assay is described in Example 1. Other suitable CGRP receptor activation assays are described in Aiyar et al., Molecular and Cellular Biochemistry, Vol. 197:179-185, 1999; Pin et al., European Journal of Pharmacology, Vol. 577:7-16, 2007; U.S. Patent No. 8,168,592, and WO 2010 / 075238, all of which are hereby incorporated by reference in their entireties.

[0073] The present invention includes pharmaceutical formulations comprising any one of the erenumab compositions described herein and one or more pharmaceutically acceptable excipients. "Pharmaceutically acceptable" refers to molecules, compounds, and compositions that are not toxic to human recipients at the dosages and concentrations used and / or do not produce allergic or adverse reactions when administered to humans. In certain embodiments, pharmaceutical formulations may contain materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the erenumab composition.In such embodiments, suitable materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as acetate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (sodium preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxaparl, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.); diluents; excipients and / or pharmaceutical adjuvants. Methods and suitable materials for formulating molecules for therapeutic use are known in the pharmaceutical art and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0074] In some embodiments, pharmaceutical formulations of the present invention comprise an erenumab composition described herein, a buffer that maintains the pH of the solution within the range of about 4.5 to about 6.5, a stabilizer, and optionally a surfactant. Suitable buffers include, but are not limited to, glutamate, acetate, Tris, citrate, histidine, succinate, and phosphate buffers. In certain embodiments, the pharmaceutical formulation comprises an acetate buffer. Acetate buffers can be made from acetic acid or acetate salts, such as sodium acetate. Other salts, such as potassium, ammonium, calcium, or magnesium acetate, can also be used. Pharmaceutical formulations containing acetate buffers typically have a pH of about 4.5 to about 5.5, or about 4.8 to about 5.2, including about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, and about 5.5.

[0075] A stabilizer refers to an excipient that stabilizes the native conformation of an antibody and / or prevents or reduces physical or chemical degradation of the antibody. Suitable stabilizers include, but are not limited to, polyols (e.g., sorbitol, glycerol, mannitol, xylitol, maltitol, lactitol, erythritol, and threitol), sugars (e.g., fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose, maltose, sucrose, trehalose, sorbose, sucralose, melezitose, and raffinose), and amino acids (e.g., glycine, methionine, proline, lysine, arginine, histidine, or glutamic acid). In some embodiments, the pharmaceutical formulation comprises a sugar as a stabilizer. In these and other embodiments, the sugar is sucrose.

[0076] In certain embodiments, the pharmaceutical formulation contains a surfactant. A surfactant is a substance that functions to reduce the surface tension of the liquid in which it is dissolved. Surfactants can be included in pharmaceutical formulations for various purposes, including, for example, preventing or controlling aggregation, particle formation, and / or surface adsorption in liquid formulations, or preventing or controlling these phenomena during lyophilization and / or the reconstitution process in lyophilized formulations. Surfactants include, for example, amphiphilic organic compounds that exhibit particle solubility in both organic solvents and aqueous solutions. Common characteristics of surfactants include their ability to reduce the surface tension of water, reduce the interfacial tension between oil and water, and also form micelles. Surfactants that can be incorporated into the pharmaceutical formulations of the present invention include both nonionic and ionic surfactants. Suitable nonionic surfactants include, but are not limited to, alkyl poly(ethylene oxide), alkyl polyglucosides such as octyl glucoside and decyl maltoside, fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific examples of nonionic surfactants include polysorbates, including, for example, polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and the like; poloxamer 188, poloxamer 407, or polyethylene-polypropylene glycol, also known as poloxalcol or poly(ethylene oxide)-poly(propylene oxide), and polyethylene glycol (PEG). Suitable ionic surfactants include, for example, anionic, cationic, and zwitterionic surfactants. Anionic surfactants include, but are not limited to, soaps, fatty acid salts, sulfonate-based or carboxylate-based surfactants, such as sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfates.Cationic surfactants include, but are not limited to, quaternary ammonium surfactants such as cetyltrimethylammonium bromide (CTAB), other alkyltrimethylammonium salts, cetylpyridinium chloride, polyethoxylated tallow amine (POEA), and benzalkonium chloride. Zwitterionic or amphoteric surfactants include, for example, dodecylbetaine, dodecyldimethylamine oxide, cocamidopropyl betaine, and cocoamphoglycine salts. In certain embodiments, the pharmaceutical formulation includes a nonionic surfactant. In one embodiment, the nonionic surfactant is polysorbate 20. In another embodiment, the nonionic surfactant is polysorbate 80.

[0077] In certain embodiments, the pharmaceutical formulation comprises an erenumab composition described herein (e.g., an erenumab composition of about 70 mg / mL to about 140 mg / mL), about 20 mM to about 40 mM acetate, about 6% to about 9% (w / v) sucrose, and about 0.008% to about 0.012% (w / v) of either polysorbate 80 or polysorbate 20. The pH of these formulations ranges from about 4.9 to about 5.5 (e.g., a pH of about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, or about 5.4). In one specific embodiment, the pharmaceutical formulation comprises 70 mg / mL of an erenumab composition described herein, about 25 mM acetate, about 7.3% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80, and the pharmaceutical formulation has a pH of about 5.2±0.2. In another specific embodiment, the pharmaceutical formulation comprises 140 mg / mL of an erenumab composition described herein, about 34 mM acetate, about 6.5% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80, and the pharmaceutical formulation has a pH of about 5.2±0.2.

[0078] Preferably, the pharmaceutical formulation is suitable for parenteral injection (e.g., intravenous or subcutaneous injection). Typical pharmaceutical forms suitable for parenteral injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Preferably, the pharmaceutical formulation is sterile and sufficiently fluid to permit delivery through a syringe or other injection device (i.e., the formulation is not excessively viscous so as to prevent passage through a syringe or other injection device). Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this filtration method can be performed either before or after lyophilization and reconstitution. Pharmaceutical compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral formulations may be placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Parenteral formulations can be stored in a syringe, autoinjector, or pen injection device or a cartridge adapted for use with such an injection device.

[0079] The above-mentioned pharmaceutical preparations may be filled into vials, syringes, automatic injection devices, or other containers or delivery devices, and may be packaged, if necessary, with instructions for use to prepare a pharmaceutical product (e.g., prescribing information including instructions for using the pharmaceutical preparation to treat, prevent, or reduce the occurrence of headaches, e.g., migraines). In certain embodiments, the pharmaceutical preparations described herein are incorporated into self-administration injection devices. Such devices are commercially available and include, but are not limited to, automatic injection devices, administration pens, microinjection pumps, and pre-filled syringes. Exemplary devices into which the pharmaceutical formulations of the present invention can be incorporated include autoinjectors (e.g., SureClick®, EverGentle®, Avanti®, DosePro®, Molly®, and Leva®), pen injection devices (e.g., Madie® pen injector, DCP™ pen injector, BD Vystra™ disposable pen, BD™ reusable pen), and prefilled syringes (BD Sterifill™, BD Hypak™, prefilled syringes from Baxter). In some embodiments, the pharmaceutical formulation is incorporated into a syringe and stored to produce a prefilled syringe. In other embodiments, the pharmaceutical formulation is incorporated into an autoinjector. The injection volume of the prefilled syringe or autoinjector can be about 2 mL or less, about 1.5 mL or less, or about 1 mL or less. In certain embodiments, the pharmaceutical formulations described herein are incorporated into a syringe or autoinjector with an injection volume of about 1 mL.

[0080] Thus, in some embodiments, the invention provides a pre-filled syringe comprising about 70 mg / mL to about 140 mg / mL of an erenumab composition described herein at a pH of about 4.5 to about 5.5, about 20 mM to about 40 mM acetate, about 6% to about 9% (w / v) sucrose, and about 0.008% to about 0.012% (w / v) polysorbate 80 or polysorbate 20. In one embodiment, the pre-filled syringe comprises a 70 mg / mL erenumab composition described herein at a pH of about 5.2±0.2, about 25 mM acetate, about 7.3% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80. In another embodiment, the pre-filled syringe contains 140 mg / mL of an erenumab composition described herein at a pH of about 5.2±0.2, about 34 mM acetate, about 6.5% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80. In any of these embodiments, the injection volume of the pre-filled syringe can be about 1 mL.

[0081] In other embodiments, the invention provides an autoinjector comprising about 70 mg / mL to about 140 mg / mL of an erenumab composition described herein at a pH of about 4.5 to about 5.5, about 20 mM to about 40 mM acetate, about 6% to about 9% (w / v) sucrose, and about 0.008% to about 0.012% (w / v) polysorbate 80 or polysorbate 20. In one embodiment, the autoinjector comprises a 70 mg / mL erenumab composition described herein at a pH of about 5.2±0.2, about 25 mM acetate, about 7.3% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80. In another embodiment, the autoinjector comprises 140 mg / mL of an erenumab composition described herein at a pH of about 5.2±0.2, about 34 mM acetate, about 6.5% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80. In any of these embodiments, the injection volume of the autoinjector can be about 1 mL.

[0082] The erenumab compositions described herein and pharmaceutical formulations comprising such compositions can be used to treat, prevent, or reduce the incidence of headaches. Accordingly, the present invention includes methods for treating, preventing, or reducing the incidence of headaches in a patient in need thereof, comprising administering to the patient any of the erenumab compositions or pharmaceutical formulations comprising such compositions described herein. In certain embodiments, the present invention provides the erenumab compositions or pharmaceutical formulations comprising the erenumab compositions described herein for use in treating, preventing, or reducing the incidence of headaches in a patient in need thereof. In other embodiments, the present invention includes the use of the erenumab compositions or pharmaceutical formulations comprising the erenumab compositions described herein in the manufacture of a medicament for treating, preventing, or reducing the incidence of headaches in a patient in need thereof. "Preventing or reducing the incidence of headaches" refers to a reduction in the frequency, duration, or severity of headaches compared to the frequency, duration, or severity of headaches before administration of the composition / formulation, or compared to the frequency, duration, or severity of headaches in a patient to whom the composition / formulation has not been administered (i.e., a control subject).

[0083] In certain embodiments, the methods or uses of the present invention treat, prevent, or reduce the occurrence of migraine headaches in a patient in need thereof by administering to the patient either an erenumab composition or a pharmaceutical formulation comprising an erenumab composition described herein. A "migraine" is a headache associated with nausea or vomiting or sensitivity to light or sound and / or characterized by at least two of the following pain characteristics: unilateral pain, throbbing pain, moderate to severe pain intensity, or pain worsened by physical activity. According to some embodiments, a pharmaceutical formulation comprising 70 mg or 140 mg of an erenumab composition described herein is administered to the patient once a month to treat, prevent, or reduce the occurrence of migraine headaches in the patient. In such embodiments, the pharmaceutical formulation may be delivered by subcutaneous injection, for example, using one of the injection devices described above (e.g., a prefilled syringe or an autoinjector).

[0084] In some embodiments, patients treated by the methods of the present invention have, suffer from, or have been diagnosed with episodic migraine. A patient with a migraine history (e.g., at least five previous migraine attacks) is diagnosed with episodic migraine if they have 14 or fewer migraine days per month. A "migraine day" includes any calendar day on which a patient experiences the onset, continuation, or recurrence of a "migraine headache" lasting more than 30 minutes, with or without aura. In some embodiments, patients with, suffer from, or diagnosed with episodic migraine have, on average, at least 4 but fewer than 15 migraine days per month. In related embodiments, patients with, suffer from, or diagnosed with episodic migraine have, on average, fewer than 15 headache days per month. As used herein, a "headache day" is any calendar day on which a patient experiences a migraine or any headache lasting more than 30 minutes or requiring acute headache treatment. In certain embodiments, a patient may be classified as having or suffering from high-frequency episodic migraine. High-frequency episodic migraine patients are those who have 8 to 14 migraine days per month. In other embodiments, patients may be classified as having or suffering from infrequent episodic migraine. Infrequent episodic migraine patients are those who have fewer than 8 migraine days per month.

[0085] In some embodiments, patients treated by the methods of the invention have, suffer from, or have been diagnosed with chronic migraine. Chronic migraine is diagnosed when a patient with migraine (i.e., a patient with at least five previous migraine attacks) has 15 or more headache days per month and at least 8 headache days are migraine days. In some embodiments, patients with, suffer from, or have been diagnosed with chronic migraine have, on average, 15 or more headache days per month.

[0086] In certain embodiments, migraine patients treated by the methods of the invention have not previously received a preventive migraine therapy. In other embodiments, migraine patients treated by the methods of the invention have failed or are intolerant to one or more preventive migraine therapies. In one such embodiment, the patient has failed to respond to at least one previous treatment with a migraine preventive drug. "Failure to respond" or "treatment failure" refers to the lack of effectiveness of a preventive drug in reducing the frequency, duration, and / or severity of migraines in a patient after a standard treatment regimen of the drug. For example, in one embodiment, a patient who has failed previous treatment with a migraine preventive drug is one who experiences the same or more migraine days per month after administration of the migraine preventive drug compared to the number of migraine days per month before treatment with the drug. Failure to respond to previous treatment with a migraine preventive drug can also include failure to tolerate the migraine preventive drug. For example, in some embodiments, a patient who has failed previous treatment with a migraine preventive drug is one who has failed to tolerate the side effects associated with the drug. In such embodiments, the side effects associated with the drug may worsen or be incompatible with another medical condition the patient has. In certain embodiments, the patient has failed or cannot tolerate treatment with one or more drugs selected from beta-blockers (e.g., propranolol, timolol, atenolol, metoprolol, and nadolol), antiepileptic drugs (e.g., divalproex, sodium valproate, valproic acid, topiramate, and gabapentin), tricyclic antidepressants (e.g., amitriptyline, nortriptyline, doxepin, and fluoxetine), and onabotulinumtoxinA.

[0087] The erenumab compositions described herein and pharmaceutical formulations containing such compositions can also be used to treat, prevent, or reduce the occurrence of other types of headache disorders, such as tension-type headache, cluster headache, hemiplegic migraine, menstrual migraine, and retinal migraine. Other diseases or conditions associated with CGRP / CGRP receptor signaling can also be treated or ameliorated by the erenumab compositions described herein and pharmaceutical formulations containing such compositions. Diseases or conditions associated with CGRP / CGRP receptor signaling include, but are not limited to, chronic pain (e.g., nociceptive pain, neuropathic pain, inflammatory pain, fibromyalgia, arthritis pain), allodynia, inflammation (e.g., neuropathic inflammation, psoriasis, osteoarthritis), type II diabetes, overactive bladder, and asthma.

[0088] The pharmaceutical preparation of the present invention is preferably administered to patients parenterally.Parenteral administration includes intraperitoneal, intramuscular, intravenous, intraarterial, intradermal, subcutaneous, intracerebral, intraventricular and intrathecal administration.In some embodiments, the pharmaceutical preparation is administered intravenously to patients.In other embodiments, the pharmaceutical preparation is administered subcutaneously to patients, for example, by subcutaneous injection.Injection can be delivered to patients using one or more of the devices (for example, pre-filled syringe and automatic injection device) described herein.

[0089] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims. [Example]

[0090] Example 1. Identification and Characterization of Erenumab Charge Variants by HIC-HPLC Aimovig is a fully human monoclonal antibody of the IgG2 subclass. The antibody is recombinantly produced in Chinese hamster ovary (CHO) cells and consists of two heavy chains and two light chains of the lambda subclass. Each heavy chain contains 456 amino acids with four intrachain disulfides. Each light chain contains 216 amino acids with two intrachain disulfides. Aimovig contains six interchain disulfides for a total of 18 intrachain and interchain disulfide bonds. The amino acid sequences for Aimovig's heavy and light chains are shown in Figures 1A and 1B, respectively. Each heavy chain contains an N-linked glycan at the consensus glycosylation site of the asparagine residue at position 306 of SEQ ID NO: 1. As is frequently observed with antibodies produced by mammalian cells, the C-terminal lysine residue at position 456 in the heavy chain is largely removed by carboxypeptidases present during production in cell culture. In addition, the N-terminal glutamine residues in both the heavy and light chains are frequently converted to pyroglutamic acid during production. The amino acid sequences for the heavy and light chains of Aimovig with these N- and C-terminal modifications are shown in Figures 1C and 1D, respectively. The calculated mass of deglycosylated Aimovig with the C-terminal lysine removed from both heavy chains and the N-terminal pyroglutamic acid structure of the intact heavy and light chains is 145,872 daltons.

[0091] Aimovig specifically binds to the extracellular domain of the calcitonin gene-related peptide (CGRP) receptor, preventing CGRP from binding to and activating the receptor. CGRP is a neuropeptide that regulates nociceptive signaling and is a vasodilator that has been implicated in the pathophysiology of migraine. Aimovig potently and specifically competes with CGRP binding to the CGRP receptor, inhibiting CGRP-induced activation of the intracellular cyclic adenosine monophosphate (cAMP) signaling cascade. Aimovig does not exhibit any significant pharmacological activity at adrenomedullin receptors, calcitonin receptors, or amylin receptors, and lacks agonist activity at the CGRP receptor.

[0092] Biochemical, biophysical, and biological characterization of erenumab drug substance produced by a commercial-scale manufacturing process was performed to elucidate the structure and functional properties of the drug substance. This example describes the identification and characterization of erenumab charge variants that exhibited reduced CGRP receptor inhibitory function. In this example, the charge heterogeneity of erenumab was evaluated by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

[0093] HIC-HPLC separates proteins primarily based on the surface hydrophobicity of the molecules, which may be affected by structural heterogeneity and other modifications that affect molecular interactions with the column matrix. Peak elution in HIC-HPLC is a function of net surface hydrophobicity, with molecules with relatively high surface hydrophobicity eluting later than molecules with relatively low surface hydrophobicity.

[0094] A sample of erenumab drug substance was loaded onto a hydrophobic interaction chromatography column (ProPac HIC-10, 5 μm particle size, 4.6 mm × 250 mm, ThermoFisher Scientific). Mobile phase A contained 10 mM sodium acetate, 1 M ammonium sulfate, pH 5.5, and mobile phase B consisted of 10 mM sodium acetate, pH 5.5. Proteins were separated using a decreasing salt gradient generated from 25% to 100% mobile phase B from 0 to 52 min, and then back to 25% mobile phase B from 55.5 to 75 min. The eluate was monitored by UV absorbance at 280 nm. The column was operated at 35°C, and the mobile phase was applied to the column at a flow rate of 0.5 mL / min.

[0095] The HIC-HPLC profile contained three characteristic regions, including a pre-peak, a main peak, and a post-peak (Figures 2A and 2B). Seven fractions spanning the pre-peak, main peak, and post-peak regions were isolated. The collected fractions were reanalyzed by HIC-HPLC, demonstrating that the fractions were of sufficient purity for characterization. The HIC-HPLC profiles and purities of the isolated fractions are shown in Figure 3 and Table 1, respectively.

[0096] [Table 1]

[0097] The pre-peak, main peak, and post-peak fractions in Figure 3 and Table 1 were characterized by various analytical techniques, including size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), reduced tryptic peptide mapping by LC-MS / MS, and cell-based bioassays.

[0098] The unfractionated drug substance and seven HIC-HPLC fractions were analyzed by SE-UHPLC using a BEH200 analytical UHPLC column (1.7 μm particle size, 4.6 mm × 150 mm, Waters Corporation) and a mobile phase containing 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8. SE-UHPLC analysis revealed that all pre-peak and post-peak fractions were enriched in high-molecular-weight (HMW) species, but only the earliest pre-peak fraction (F1) and the latest post-peak fraction (F7) were enriched in low-molecular-weight (LMW) species compared to the unfractionated drug substance (Table 2).

[0099] [Table 2]

[0100] Biochemical modifications of erenumab present in the enriched pre-peak, main peak, and post-peak fractions, as well as in the unfractionated drug substance, were assessed by reduced tryptic peptide mapping with detection by electrospray ionization tandem mass spectrometry (ESI-MS / MS). The drug substance and seven HIC-HPLC fractions were denatured in 7.5 M guanidine hydrochloride solution, treated with 0.5 M dithiothreitol (DTT), and all cysteine ​​residues were alkylated by adding 0.5 M sodium iodoacetate (IAA). The reduced and alkylated samples were desalted by gel filtration and then digested with trypsin for 35 minutes at 37°C. The digested samples were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (1.8 μm particle size, 2.1 mm × 150 mm) and a 0.1% TFA mobile phase with an acetonitrile gradient at a flow rate of 0.3 mL / min. Peptide detection was by ultraviolet (UV) light absorbance at 215 nm, followed by peak identification by online ESI-MS / MS. The fragmentation pattern of each peptide ion was then examined against its predicted MS / MS spectrum.

[0101] An overlay of the bulk and enriched fractions is shown in Figure 4, showing the elution regions for two peptides within heavy chain CDR3: TH12H13 (amino acid residues 99-113 of SEQ ID NO: 1) and TH13 (amino acid residues 101-113 of SEQ ID NO: 1). Pre-peak fractions F2, F3, and F4 and post-peak fraction F6 all showed diversity in the CDR3 peptide (TH13) relative to the bulk. No differences in the TH13 peptide were observed for fractions F1 and F7. Pre-peak fractions F2, F3, and F4 and post-peak fraction F6 were enriched in both deamidated and isomerized variants (isoaspartic acid (IsoAsp) and succinimide intermediates) within the heavy chain CDR3 peptide TH13. Deamidation of the asparagine residue at position 102 and isomerization of the aspartic acid residue at position 105 (both positions relative to SEQ ID NO: 1) were observed. Two separate peptides identified as IsoAsp105 were observed eluting at different retention times. These peptides may represent structural enantiomers generated during aspartic acid isomerization. An additional TH13 peptide variant corresponding to a succinimide intermediate at position 105 was also observed eluting after the native peptide. The modification level was approximated by comparing the peak areas of the extracted ion chromatograms (EICs) of the modified peptides with those generated from the unmodified peptide. Mass spectrometry confirmed the presence of increased deamidation and IsoAsp variants within the pre- and post-peak regions (Table 3).

[0102] [Table 3]

[0103] Pre-peak fraction F1 was reduced and analyzed by ESI-TOF mass spectrometry because previous analysis of this fraction by rCE-SDS showed significant levels of LMW and medium molecular weight (MMW) species. A total of six cleavage fragments were identified in pre-peak fraction F1, including four cleavage sites (Leu). 315 / Thr 316 ;Ser 197 / Val 198;Leu 195 / Ser 196 and Leu 192 / Tyr 193 ) were located within the heavy chain CH1 and CH2 domains. 108 / Tyr 109 and Asp 105 / Ser 106 ) was also identified in the CDR3 of the heavy chain. For all species, only the C-terminal fragment was detected. Leu 195 / Ser 196 Elevated levels of cleavage sites were observed within pre-peak fraction F1 relative to the drug substance (Table 3).

[0104] The bioactivity of the enriched HIC-HPLC fraction compared to erenumab bulk was evaluated by a cell-based bioassay. The cell-based bioassay assesses erenumab's potency by measuring its ability to inhibit ligand-induced activation of the human CGRP receptor. CGRP receptors are G protein-coupled receptors, and receptors of this family have been shown to generate cAMP intracellularly as part of their signaling mechanism. A stable Chinese hamster ovary K1 (CHO-K1) cell line expressing the human CGRP receptor (CHO-K1 huCGRP) was incubated with CGRP ligands and various concentrations of erenumab standard and test samples. The amount of cAMP produced by cells after incubation with CGRP in the presence or absence of erenumab standard and test samples was measured using a competitive homogeneous time-resolved fluorescence energy transfer (TR-FRET) assay, in which a detection signal was generated when the labeled assay components (Alexa Fluor® 647 dye-labeled cAMP and anti-cAMP monoclonal antibody-cryptate) bound to each other. When cAMP was generated intracellularly by CGRP receptor activation, the natural cAMP produced by the cells competed with Alexa Fluor® 647 dye-labeled cAMP for binding to the cryptate-labeled anti-cAMP monoclonal antibody, reducing the detection signal. Therefore, due to the competitive nature of the TR-FRET cAMP assay, the signal generated is inversely proportional to the concentration of cAMP in the cells. The TR-FRET signal was measured using a plate reader. The activity of the test sample was determined by comparing the signal generated by the test sample with the signal generated by the erenumab standard and reported as relative potency.

[0105] As shown in Table 4 below, all pre-peak fractions showed significant reductions in potency. These reductions were due to either high levels of fragmentation (for fraction F1) or deamidation and isomerization in the heavy chain CDR3 region (for fractions F2-F4), which are predominant variants in the pre-peak fractions as described above. The post-peak fractions also showed increased HMW species and low levels of heavy chain CDR3 aspartic acid (Asp), which are predominant variants in the post-peak fractions as described above. 105 ) showed a reduction in potency that may be the result of isomerization.

[0106] [Table 4]

[0107] The results of the analysis described in this example demonstrated that the HIC-HPLC profile of erenumab drug substance contains three distinct regions, including a pre-peak, a main peak, and a post-peak. The major erenumab variants detected by HIC-HPLC include heavy chain CDR3 aspartic acid isomerization (e.g., Asp in SEQ ID NO: 1) in both the pre-peak and post-peak regions. 105 isomerization of Asn in SEQ ID NO: 1) and deamidation (e.g., 102 These include fragmented species (deamidated variants of HMW species). Fragmented species were observed in the pre-peak group, and elevated levels of HMW species were observed in the post-peak group. The potencies of all erenumab HIC-HPLC pre-peak and post-peak fractions were lower than that of the drug substance, as assessed by cell-based bioassays. The pre-peak showed a significant reduction in potency as a result of aspartic acid isomerization and deamidation in the heavy chain CDR3 region and a high level of fragmented variants. The post-peak also showed a reduction in potency as a result of a high level of HMW species and heavy chain CDR3 isomerized variants.

[0108] Because the presence of aspartic acid isomerization and asparagine deamidation variants in the heavy chain CDR3 of Aimovig affected the inhibitory potency of the composition, several lots of Aimovig manufactured on a commercial scale (140 mg / mL) were analyzed by HIC-HPLC to assess the presence and amount of isomerization and deamidation variants, as measured by the percentage of peak area of ​​the pre-peak in the HIC-HPLC chromatogram. The potency of the drug substance lots was also evaluated by the cell-based potency assay described above and compared to the potency of lot number 78137, the Aimovig drug substance sample employed in the Phase II / Phase III clinical trial. A summary of the data is provided in Table 5 below.

[0109] [Table 5]

[0110] As shown by the data in Table 5, commercial-scale erenumab drug substance contained consistent levels of isomerized and deamidated variants ranging from 1.7% to 2.1%, as measured by HIC-HPLC pre-peak. Drug substances containing levels of deamidated / isomerized variants in this range exhibited potency comparable to that of the erenumab drug substance employed in clinical trials.

[0111] Example 2. Evaluation of Erenumab isomerization and deamidation under various storage conditions Asn 102 Deamidation of Asp 105 Conversion of Asn to isoaspartic acid was observed in CDR3 of the erenumab heavy chain during the manufacture of the drug substance (Example 1). Erenumab variants with these modifications exhibited reduced potency compared to the unmodified form of erenumab (Example 1). The effect of Asn on the potency of erenumab drug substance 102 Deamidation and Asp 105To further evaluate the impact of isomerization variants, Aimovig was subjected to stress conditions to increase the formation of deamidated and isomerized variants, and the efficacy of the stressed drug substance was assessed. Specifically, Aimovig was subjected to the following three stress conditions to facilitate the generation of deamidated and isomerized variants: Heat exposure (14 days at 50°C) Physiological pH and temperature (pH 7.4 at 37°C for 14 days) High pH exposure (pH 8.0 at 25°C for 14 days)

[0112] The formation of deamidated and isomerized variants induced by each stress condition over time was monitored by HIC-HPLC and reduced tryptic peptide mapping methods as described in Example 1. For each stress condition, samples were removed at various time points throughout the duration of the test and freezing. After the final time point, all samples were analyzed in parallel to minimize analytical variability.

[0113] heat exposure The results of HIC-HPLC analysis of erenumab drug substance stressed at 50°C showed a significant increase in the pre-peak (Figure 5 and Table 6). This increase in the pre-peak is primarily due to increased aspartic acid isomerization after 14 days of exposure to heat stress, as revealed by reduced tryptic peptide mapping analysis described in detail below. An increase in the post-peak was also observed, likely due to increased levels of HMW species, as these eluted as part of the relatively later-eluting post-peak, as shown in Table 2 in Example 1.

[0114] [Table 6]

[0115] Peptide map chromatograms were evaluated for the presence of new peaks or significant changes in peak area of ​​existing peaks over the exposure time. A magnified scale overlay of the day 0 and day 14 samples highlighting the areas where differences were observed is shown in Figure 6. Asp in heavy chain CDR3 (peptides TH12H13 and TH13) 105 Aspartic acid isomerization at the Fc region was the major degradation product observed in the peptide map overlay. 315 and Thr 316 A hydrolysis fragment of peptide TH26 (corresponding to amino acid residues 311 to 326 of SEQ ID NO: 1) between was also observed in the chromatogram of the 14 day sample.

[0116] The biological activity of erenumab drug substance exposed to heat stress conditions was evaluated by the cell-based bioassay described in Example 1. As shown in Table 7, heat stress negatively affected the relative potency of erenumab drug substance after exposure to 50°C for 14 days. The reduction in potency after heat stress was due to the Asp 105 This was attributed to an increase in isomerized variants and high molecular weight species, both of which were shown to affect potency (see Examples 1 and 5).

[0117] [Table 7]

[0118] Physiological pH and temperature Aimovig was exposed to physiological pH by dilution to approximately 10 mg / mL with phosphate-buffered saline (PBS) solution at pH 7.4 and incubated at 37°C (physiological temperature) for 14 days. The pH control, which was drug substance diluted in formulation buffer (15 mM sodium acetate, 8.2% (w / v) sucrose, 0.010% (w / v) polysorbate 80), pH 5.2, was incubated at 37°C for 14 days. Thus, the "physiological pH stressed samples" were exposed to both pH and heat stress, while the "pH control samples" were exposed to heat stress only.

[0119] Comparison of the HIC-HPLC profiles for the day 14 physiological pH stressed samples and the day 0 samples showed an increase in both the pre- and post-peak (Figure 7 and Table 8). The day 14 traces from the physiological pH stressed and pH control samples showed similar levels of the pre-peak eluting at approximately 30 minutes, which was due to the presence of Asp. 105 This corresponds to an isomerized variant, suggesting that this isoaspartic acid variant is primarily promoted by heat stress rather than pH stress. 102 The pre-peak eluting at approximately 34 min, corresponding to the deamidated variant, increased in the physiological pH stressed sample on day 14 but not in the pH control, which was due to the Asp 102 This indicates that deamidation of β-glucan is primarily driven by pH stress, not heat stress. An increase in the post-peak was also observed in the physiological pH-stressed samples at day 14 compared to both the day 0 and day 14 pH control samples, indicating that the change is primarily driven by pH stress.

[0120] [Table 8]

[0121] Biochemical modification of Aimovig ® (Erenumab) drug substance stressed at physiological pH and temperature was monitored by reduced peptide mapping by mass spectrometry (MS) after trypsin digestion. Analysis of MS data revealed that the heavy chain CDR3 (Asn 102 ) and Fc region (Asn 393 and Asn 398 ) and heavy chain CDR3 Asp 105 The amount of isomerization increased (data not shown). 105 Isomerization and Asn 102 The increased level of deamidation is consistent with the increase observed in the pre- and post-peak by HIC-HPLC shown in FIG.

[0122] The biological activity of erenumab drug substance stressed at physiological pH and temperature was evaluated by cell-based bioassay. As shown in Table 9, physiological pH and temperature stress resulted in a decrease in the potency of erenumab drug substance after 14 days of exposure. 102 Deamidation and Asp 105 Although increased levels of isomerized variants were observed under these stress conditions, only a slight reduction in potency was observed, which may be due to the overall levels of these deamidated and isomerized variants, which accounted for approximately 6% of the drug substance, being too low to significantly affect potency as measured by cell-based bioassays.

[0123] [Table 9]

[0124] high pH exposure Aimovig drug substance was exposed to high pH by dilution to approximately 10 mg / mL with Tris base solution at pH 8.0 and incubated for 14 days at 25° C. The pH control, which was drug substance diluted in formulation buffer (15 mM sodium acetate, 8.2% (w / v) sucrose, 0.010% (w / v) polysorbate 80), pH 5.2, was incubated for 14 days at 25° C.

[0125] The results of HIC-HPLC analysis showed that Asn was significantly higher than that of the day 0 sample and the pH control sample. 102 There was an increase in the pre-peak due to deamidation and an increase in the post-peak in the high pH stressed samples at day 14 (Figure 8 and Table 10).

[0126] [Table 10]

[0127] Biochemical modification of Aimovig under high pH stress was monitored by reduced peptide mapping with MS after trypsin digestion. Similar to physiological pH stress conditions, Asn 102 Heavy chain CDR3 and Asn 393 and Asn 398 An increase in deamidation in the Fc region at 14 days was observed for stressed samples compared to day 0 and pH control samples. The biological activity of erenumab drug substance stressed at high pH was evaluated by cell-based bioassay. As shown in Table 11, high pH stress moderately affected the potency of erenumab drug substance after 14 days of exposure. Similar to the results obtained under physiological pH and temperature stress conditions, the Asn present in the stressed drug substance 102 The overall levels of deamidated variants were too low to significantly affect potency, as measured by cell-based bioassays.

[0128] [Table 11]

[0129] Asn required to significantly reduce the efficacy of erenumab drug substance 102 Deamidation and Asp 105 To better identify the levels of isomerization variants, statistical analyses modeling the relationship between potency and percentage of HIC-HPLC pre-peak area were performed on the available data from various Ereneuma blots stored at different temperatures. Ereneuma blots were stored at 5°C (13 lots), 25°C (4 lots), 30°C (4 lots), or 40°C (5 lots). For data from the 5°C storage condition, there were 6 lots with only one time point and 9 lots with only two time points. All other storage conditions had data from at least three different time points.

[0130] The percent relative potency, as measured by cell-based bioassay, was plotted as a function of the percent peak area for the pre-peak in the HIC-HPLC chromatograms for the four different storage conditions, and a regression analysis was performed ( FIG. 9 ). An estimate of the slope of the fitted regression line was determined for each temperature, along with an adjusted p-value for the test, to determine whether the slope was different from zero (Table 12). The slope of the regression line for the data from the 40° C. storage condition was statistically significant from zero at the 0.05 significance level. Based on the regression analysis of the data from the 40° C. storage condition, a 1% increase in the HIC-HPLC pre-peak area resulted in a 1.14% decrease in relative potency.

[0131] [Table 12]

[0132] The relative potency of the erenumab drug substance employed in clinical trials exceeded 70% as measured by cell-based bioassays. Asn that would reduce the potency of erenumab drug substance below the level acceptable for clinical trials (70%) 102 Deamidation and Asp 105 To estimate the level of isomerized variants (represented by the HIC-HPLC pre-peak), the fitted regression line for the 40°C storage condition shown in Figure 9 was extrapolated to show the % area of ​​the HIC-HPLC pre-peak for a relative potency of 70%. As shown in Figure 10, the predicted value of the % HIC-HPLC pre-peak area for a relative potency of 70% based on the fitted regression line is approximately 28.85%.

[0133] The results of the experiments described in this example demonstrate that Asn in erenumab bulk drug 102 Levels of deamidated variants and / or Asp 105It has been demonstrated that an increase in the level of isomerized variants results in a decrease in the ability of erenumab to inhibit CGRP-induced activation of the CGRP receptor. The level of deamidated and isomerized variants should be monitored and controlled to less than about 30% of the drug substance to maintain the efficacy of the drug substance at a level similar to that of the drug substance used in clinical trials. In some embodiments, Asn in the drug substance 102 Deamidation and / or Asp 105 A relatively low level of isomerization variants (e.g., less than about 15%) is desirable because about 17% of these variants in the drug substance resulted in a significant reduction in the potency of erenumab drug substance (see Tables 6 and 7).

[0134] Example 3. Identification and characterization of Erenumab charge variants by CEX-HPLC This example describes the identification and characterization of additional charge variants of erenumab that exhibited reduced CGRP receptor inhibitory function. The charge heterogeneity of erenumab in this example was assessed by cation exchange high performance liquid chromatography (CEX-HPLC).

[0135] CEX-HPLC separates proteins primarily based on surface charge heterogeneity; however, this may be influenced by structural heterogeneity and other modifications that affect molecular interactions with the ion-exchange resin. Peak elution in this method is a function of the net surface charge, with early-eluting negatively charged species and late-eluting positively charged species.

[0136] A sample of erenumab drug substance was loaded onto an analytical CEX-HPLC column (BioPro SP-F, 5 μm particle size, 4.6 mm × 100 mm, YMC America, Inc.). Mobile phase A contained 20 mM sodium phosphate at pH 6.6, and mobile phase B consisted of 20 mM sodium phosphate at pH 6.6, 500 mM sodium chloride. Proteins were separated using a linear salt gradient generated from 5% to 12% mobile phase B from 0 to 4 min, 23% mobile phase B at 18 min, 100% mobile phase B from 18.5 to 20.5 min, and then back to 5% mobile phase B from 21 to 25 min. The eluate was monitored by UV absorbance at 280 nm. The column was operated at 28 °C, and the mobile phase was applied to the column at a flow rate of 0.6 mL / min.

[0137] The CEX-HPLC profile contained three characteristic regions, including an acidic peak, a main peak, and a basic peak (Figure 11). Nine fractions spanning the acidic, main, and basic peak regions were isolated. The collected fractions were reanalyzed by CEX-HPLC, and the fractions were demonstrated to be of sufficient purity for characterization. The CEX-HPLC profiles and purities of the isolated fractions are shown in Figure 12 and Table 13, respectively.

[0138] [Table 13]

[0139] The biological activity of each of the enriched CEX-HPLC fractions compared to erenumab drug substance was evaluated by the cell-based bioassay described in Example 1. As shown in Table 14, both acidic peak fractions F1 and F2 showed reduced potency compared to drug substance, while the remaining acidic, main, and basic peak fractions showed no significant differences.

[0140] [Table 14]

[0141] To more fully understand which variants in the acidic fraction had an effect on potency, the concentrated CEX-HPLC fractions were characterized by various analytical techniques, including non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), reducing sodium dodecyl sulfate capillary electrophoresis (rCE-SDS), and non-reducing reverse-phase high-performance liquid chromatography (RP-HPLC).

[0142] Unfractionated drug substance and nine CEX-HPLC fractions were analyzed by nrCE-SDS. Samples were denatured by heating in the presence of sodium dodecyl sulfate (SDS) and N-ethylmaleimide at pH 6.5 before electrokinetic injection into a bare fused silica capillary filled with SDS gel buffer at 25°C. Absorbance was monitored at 220 nm. As shown in Table 15 below, acidic fraction F1 was enriched in both pre-peak and post-peak species compared to drug substance. The pre-peak species contained species with lower molecular weights than intact erenumab (main peak) and resulted from either peptide hydrolysis or partial molecular association, while the post-peak species were larger in size compared to the main peak. Overall, the relative amounts of pre-peak species were consistent among the remaining acidic, main, and basic peak fractions (i.e., F2 to F9) and drug substance, although slight differences in distribution within the pre-peak group were observed.

[0143] [Table 15]

[0144] Aimovig drug substance and nine CEX-HPLC fractions were also analyzed by rCE-SDS. The method for rCE-SDS was similar to that for nrCE-SDS, except that the sample was reduced and denatured by heating in the presence of SDS and β-mercaptoethanol before injection into the capillary. The results of rCE-SDS analysis showed that all fractions, except for the acidic peak fraction F1, were similar to the unfractionated drug substance control. The acidic peak fraction F1 was significantly enriched in LMW and MMW species, which most likely correspond to peptide hydrolysis fragments (data not shown). The acidic peak fraction F1 and, to a lesser extent, fraction F2, were enriched after the heavy chain, which corresponds to non-consensus glycosylation variants.

[0145] The drug substance and CEX-HPLC fractions were then analyzed by RP-HPLC using a Waters BEH300 C4 column (1.7 μm particle size, 2.1 mm × 50 mm) and eluted with a 0.1% TFA-containing mobile phase and a gradient of 1-propanol at 75°C. Absorbance at 215 nm was monitored. Across the CEX-HPLC profile, there was an observed trend toward enrichment of disulfide isoforms, with IgG2-B and IgG2-A / B isoforms enriched in acidic fractions, while the IgG2-A disulfide isoform was enriched in the main and basic peak fractions (Figure 13 and Table 16). As described in more detail in Example 4, the IgG2-B disulfide isoform was significantly less potent than erenumab drug substance or the IgG2-A and IgG2-A / B disulfide isoforms. The RP-HPLC profile of the acidic peak fraction F1 did not match the chromatogram of the drug substance, which was most likely a result of the significant levels of reducible covalent fragmentation observed with rCE-SDS and nrCE-SDS, respectively, as described above.

[0146] [Table 16]

[0147] The results of the analysis described in this example indicate that certain acidic variants of Aimovig, such as fragmentation variants and disulfide isoform variants corresponding to the acidic peak in the CEX-HPLC chromatogram, are less potent than Aimovig drug substance. In particular, acidic fraction F1 exhibited a significant reduction in potency as a result of the high level of fragmentation detected by rCE-SDS and nrCE-SDS. Acidic fraction F2 also exhibited a reduction in potency due to the increased level of IgG2-B disulfide isoforms enriched in this fraction compared to the unfractionated drug substance. Overall, the major Aimovig variants detected by CEX-HPLC include disulfide isoform variants, with IgG2-B and IgG2-A / B isoforms enriched in the acidic peak. Deamidation, fragmentation (LMW and MMW), HMW species, and non-consensus glycosylation variants were also detected in the acidic peak fraction.

[0148] Several lots of commercial-scale manufactured erenumab drug substance (140 mg / mL) were analyzed by CEX-HPLC to assess the presence and amount of acidic variants (e.g., IgG2-B disulfide isoforms) as measured by the peak area percentage of the acidic peak in the CEX-HPLC chromatogram. The potency of the drug substance lots was also evaluated by cell-based potency assays and compared to the potency of lot number 78137, the erenumab drug substance sample employed in Phase II / Phase III clinical trials. A summary of the data is provided in Table 17 below.

[0149] [Table 17]

[0150] As shown by the data in Table 17, commercial-scale manufactured erenumab drug substance contained consistent levels of acidic variants ranging from 28.7% to 31.3%, as measured by HIC-HPLC acidic peak. Drug substances containing levels of acidic variants in this range exhibited potency comparable to that of the erenumab drug substance employed in clinical trials.

[0151] Example 4. Disulfide isoform variants of Erenumab Aimovig is an antibody of the IgG2 subclass and may therefore exhibit disulfide isoform variants that have been described for IgG2 molecules (Dillon et al., 2011). (Wypych et al., J Chromatogr A., ​​Vol. 1120(1-2):112-120, 2006; Wypych et al., Journal of Biological Chemistry, Vol. 283(23):16194-16205, 2008; Dillon et al., Journal of Biological Chemistry, Vol. 283(23):16206-16215, 2008). The connectivity of disulfide bonds detected in erenumab was elucidated using non-reduced and reduced Lys-C peptide maps combined with electrospray ionization tandem mass spectrometry (ESI-MS / MS) for identification. Through this approach, predicted disulfide bonds in the classical IgG2-A structure were elucidated, as well as disulfide bonds corresponding to the IgG2-A / B and IgG2-B structures.

[0152] Disulfide-linked peptides were identified in the bulk product by peptide mapping using endoprotease Lys-C under non-reducing and reducing conditions as shown in Figure 14. The outlet of the reversed-phase high-performance liquid chromatography (RP-HPLC) separation was coupled to electrospray ionization tandem mass spectrometry (ESI-MS / MS) for orthogonal mass analysis in addition to absorbance detection. A portion of the non-reduced digest was treated with the reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and analyzed using the same RP-HPLC conditions.

[0153] The non-reduced Lys-C map (Figure 14, top trace) is labeled with peaks "A" through "G" and "a" through "i" that disappear under reducing conditions. The disappearance of these peptides after reduction indicates that they participate in disulfide bonds in the native protein. Peptides containing disulfide bonds in the non-reduced Lys-C digest and the corresponding reduced peptides in the reduced Lys-C digest were determined by their presence or absence in the absorbance chromatogram and confirmed by intact peptide mass accuracy and the presence of characteristic MS / MS product ions. The theoretical and observed masses for all of the peptide ions used to verify the disulfide-linked peptides are shown in Tables 18 and 19 for the non-reduced and reduced peptide maps, respectively. The identified non-reduced and reduced peptides illustrate the predicted IgG2-A disulfide isoform structure, shown schematically in Figure 15A.

[0154] [Table 18]

[0155] [Table 19]

[0156] Lys-C peptide mapping revealed minor disulfide-linked peptide peaks corresponding to heterogeneity in disulfide pairing between the hinge peptide and other regions of the molecule. In addition to the major disulfide-linked peptide peaks labeled in Figure 14, there were minor late-eluting peaks ("a"-"i") in the non-reduced map that were not present in the reduced map, indicating that they participate in disulfide bonds in the native protein. These late-eluting peptides were identified by mass spectrometry as interchain disulfide-linked peptides for known structural isoforms of IgG2 (Wypych et al., 2004). al., Journal of Biological Chemistry, Vol. 283(23):16194-16205, 2008; Dillon et al., Journal of Biological Chemistry, Vol. 283(23): 16206-16215, 2008; and Zhang et al., Anal Chem., Vol. 82(3):1090-1099, 2010). Peptides "a," "b," "d," and "e" correspond to the structural isoform IgG2-B, while peptides "c," "f," and "i" correspond to the structural isoform IgG2-A / B. Schematic representations of the IgG2-A / B and IgG2-B disulfide isoform structures are shown in Figures 15B and 15C, respectively. The theoretical and observed masses for all of the peptide ions used to validate the IgG2-A / B and IgG2-B disulfide-linked peptides are shown in Table 18. Analysis suggests that the disulfide bonds in erenumab are consistent with those previously observed for IgG2 antibodies (Wypych et al., 2010). al., 2008; Dillon et al., 2008; and Zhang et al., 2010).

[0157] As detailed above, peptide mapping revealed the presence of disulfide-linked peptides for the predominant IgG2-A structure, as well as peptides with binding properties associated with additional IgG2-A / B and IgG2-B disulfide structure isoforms. As shown in Figure 15, in the IgG2-B isoform, both Fab arms are linked to the hinge region, whereas in the IgG2-A isoform, neither Fab arm is linked to the hinge. The IgG2-A / B isoform is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Separation of IgG2 disulfide isoforms by non-reducing reversed-phase high-performance liquid chromatography (RP-HPLC) has been previously reported (Wypych et al., 2008 and Dillon et al., 2008). In these studies, peptides corresponding to disulfide isoforms A, A / B, and B were identified in RP-HPLC peak fractions (Wypych et al., 2008 and Dillon et al., 2008). A representative profile for erenumab drug substance analyzed by non-reduced RP-HPLC is shown in Figure 16A, with the disulfide isoform peaks labeled consistent with the elution order reported by Wypych et al., 2008. The late-eluting peak was identified as the IgG2-A isoform and is grouped with the IgG2-A peak for relative percentage reporting. The relative levels of disulfide isoforms in erenumab drug substance were approximately 59.5% IgG2-A, 34.7% IgG2-A / B, and 4.4% IgG2-B, with approximately 1.5% of the early-eluting pre-peak species.

[0158] Although non-reduced RP-HPLC is an effective method for determining the relative amounts of disulfide isoform variants, the mobile phase and separation conditions are incompatible with the cell-based bioassay used to determine the inhibitory potency of erenumab. Therefore, disulfide isoform-enriched fractions were collected from semi-preparative CEX-HPLC as described in Example 3 and tested for potency using the cell-based bioassay described in Example 1. Three CEX-HPLC fractions enriched in disulfide isoforms IgG2-A, IgG2-A / B, and IgG2-B relative to drug substance (Table 16 and Figure 13A) were analyzed for potency as described in more detail below. The enriched IgG2-A, IgG2-B, and IgG2-A / B disulfide isoform CEX-HPLC fractions were analyzed by non-reduced RP-HPLC to confirm sufficient purity prior to potency evaluation. The RP-HPLC overlay of the three CEX-HPLC collected fractions against the unfractionated drug substance is shown in FIG. 16B, and the RP-HPLC disulfide isoform distribution data is provided in Table 20.

[0159] [Table 20]

[0160] As shown in Table 21, the relative potencies of the IgG2-A and IgG2-A / B enriched isoform fractions are similar to that of erenumab bulk drug. However, the fraction enriched for the IgG2-B disulfide isoform showed significantly reduced potency compared to bulk drug and the enriched IgG2-A / B and IgG2-A isoform fractions.

[0161] [Table 21]

[0162] These data suggest that the conformation of the disulfide bond in the hinge region of Aimovig is important for the antibody's biological activity. In particular, elevated levels of the IgG2-B disulfide isoform in the bulk drug significantly impact inhibitory potency. Analysis of several lots of Aimovig manufactured at commercial scale revealed consistent levels of the IgG2-B disulfide isoform in the bulk drug, ranging from 4.6% to 5.2%, as measured by non-reduced RP-HPLC. The levels of the IgG2-A / B and IgG2-A disulfide isoforms in these Aimovig bulk drug lots were also consistent with IgG2-A / B levels ranging from 34.2% to 35.5% and IgG2-A levels ranging from 57.4% to 59.3%.

[0163] Example 5. Identification and characterization of erenumab size variants This example describes the identification and characterization of size variants of erenumab that exhibit reduced CGRP receptor inhibitory function. Size variants of erenumab can include high molecular weight (HMW) species and low molecular weight (LMW) species. HMW species, which are species larger than monomers (e.g., dimers and higher oligomeric species), can be formed by non-covalent association, reducible covalent association, and / or irreducible covalent association. LMW species can arise from fragmentation of the polypeptide backbone and / or incomplete assembly of subunit components such as light and heavy chains.

[0164] In this example, the size heterogeneity of erenumab was evaluated by SE-UHPLC. SE-UHPLC separates proteins primarily based on differences in hydrodynamic volume. The SE-UHPLC method was performed under non-reducing, non-denaturing conditions to assess the size distribution of erenumab under native conditions. A sample of erenumab drug substance was loaded onto an analytical SE-UHPLC column (BEH200 column, 1.7 μm particle size, 4.6 mm × 150 mm, Waters Corporation), and proteins were separated isocratically using a mobile phase containing 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8. The eluate was monitored by UV absorbance at 280 nm. The column was operated at ambient temperature, and the mobile phase was applied to the column at a flow rate of 0.4 mL / min.

[0165] Three distinct regions were evident in the SE-UHPLC profile of erenumab drug substance, including a predominant monomeric (main) peak and lower levels of HMW (pre-peak) and LMW (post-peak), as shown in Figures 17A and 17B. Fractions derived from the pre-peak, main peak, and post-peak regions were collected, and the variants separated in the fractions were characterized. The HMW peak by SE-UHPLC (Figure 17B) consisted of two incompletely resolved species and was therefore collected as two distinct fractions representing the leading and tailing edges of the HMW peak, respectively. The two HMW peaks and the main peak were isolated using semi-preparative SE-HPLC and then further concentrated by analytical SE-HPLC. The collected HMW and main peak fractions were concentrated using molecular weight cutoff filters and buffer-exchanged into 15 mM sodium acetate at pH 5.2 prior to characterization analysis. The LMW peak by SE-UHPLC (Figure 17B) consists of two minor peaks, the sum of which is below the assay limit of quantitation of 0.3% in drug substance. The LMW peak was isolated and concentrated using analytical SE-UHPLC and subjected to limited characterization studies as described in more detail below.

[0166] Characterization of SE-UHPLC HMW fractions The SE-UHPLC profiles and purity of the isolated HMW and main peak fractions, shown in Figure 18 and Table 22, respectively, demonstrate that the fractions were sufficiently pure for characterization.

[0167] [Table 22]

[0168] The concentrated HMW and main peak fractions in Figure 18 and Table 22 were characterized by various analytical techniques, including SE-UHPLC with static light scattering detection, reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS), non-reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), and cell-based bioassays.

[0169] The HMW and main peak fractions, along with the unfractionated drug substance, were analyzed by SE-HPLC with static light scattering detection (SE-HPLC-SLS) to determine the molar mass of the dominant peak in each chromatogram. SE-HPLC-SLS analysis was performed using an Agilent 1100 HPLC system. The column was a TSK-GEL G3000SWxl, 5 μm particle size, 7.8 mm ID x 300 mm column length (Tosho Biosep). The detectors used were a Wyatt Heleos II light scattering detector, a Wyatt Optilab rEX RI detector, and an Agilent UV detector with a wavelength setting of 280 nm. The SE-HPLC run was performed at room temperature with a 100 mM potassium phosphate, 250 mM potassium chloride, pH 6.8 buffer solution as the mobile phase, and the flow rate was 0.5 mL / min. The injection volume was 4.3 μL, and 300 μg of protein was injected. For molecular weight (MW) calculations, LS (light scattering) and RI (refractive index) signals and a sample refractive index increment (dn / dc) value of 0.185 were used.

[0170] The measured mass of the monomer (intact antibody containing two heavy chains and two light chains) from the main peak fraction was 149 kDa, and that of the bulk control was 143 kDa, both of which are consistent with the theoretical molecular mass of the erenumab monomer (148.8 kDa) (Table 23). The measured masses of the HMW peaks for both HMW-enriched fractions (HMW1 and HMW2) were 292 kDa and 279 kDa, respectively, both of which are consistent with the molecular mass of the erenumab dimer (297.6 kDa) (Table 23). These data demonstrate that the fractions enriched in both HMW1 and HMW2 are primarily composed of the erenumab dimer.

[0171] [Table 23]

[0172] Analysis of nrCE-SDS enriched fractions and unfractionated drug substance according to the method described in Example 3 revealed that both HMW fractions were enriched in a post-peak corresponding to covalently linked high molecular weight species larger than the monomer (main peak), with a pre-peak area comparable to the control (Table 24).

[0173] [Table 24]

[0174] The ratio of non-covalent to covalent dimers can be approximated by comparing the purity of fractions enriched under native conditions with that under denaturing conditions. By comparing the dimer level under native conditions (SE-UHPLC: % HMW in Table 22) with the covalent dimer level under denaturing conditions (nrCE-SDS: % post-peak in Table 24), the level of covalent dimer can be estimated in the native drug substance. Based on this comparison, the majority of the native dimer species in Aimovig drug substance are covalently bound, with approximately 68% of the HMW1 dimer species and almost all of the HMW2 dimer remaining under the denaturing test conditions.

[0175] Aimovig drug substance and the enriched HMW species and main peak fractions were also analyzed by rCE-SDS according to the method described in Example 3. As shown in Figure 19, the results of the rCE-SDS analysis indicated that both the HMW1 and HMW2 fractions were enriched in medium molecular weight (MMW) and high molecular weight (HMW) species relative to the drug substance. Similar to assessing the levels of covalent and non-covalent dimeric species, rCE-SDS can be used to approximate the levels of reducible and non-reducible dimeric species in the drug substance. Comparing the dimer level under native conditions (SE-UHPLC: % HMW) with the non-covalent dimer level under reducing and denaturing conditions (nrCE-SDS: % HMW), the non-covalent dimer level can be estimated in the native drug substance. Based on the comparison as shown in Table 25, the majority of the native dimeric species in Erenumab drug substance can be reduced, with approximately 87% of the HMW1 dimeric species and approximately 93% of the HMW2 dimeric species being reduced to their individual components under the reduction and denaturation test conditions.

[0176] [Table 25]

[0177] The bioactivity of the enriched SE-UHPLC fractions compared to erenumab drug substance was evaluated by the cell-based bioassay described in Example 1. As shown in Table 26, both the HMW1- and HMW2-enriched fractions exhibited reduced potency compared to the drug substance and the enriched main peak. A significant portion of the HMW species in these fractions are covalently linked and cannot be separated, as described above. Self-association imposes steric hindrance that can lead to conformational changes, which may affect the binding of the molecule to the CGRP receptor target, explaining the observed reduced potency.

[0178] [Table 26]

[0179] Characterization of SE-UHPLC LMW fractions The LMW peak (Figure 17B) was collected by SE-UHPLC in a single fraction and concentrated using a molecular weight cutoff filter prior to characterization analysis. Reinjection of the LMW fraction onto SE-UHPLC showed enrichment of the LMW species to approximately 34.4% (data not shown). The collected and concentrated LMW fraction was analyzed by mass spectrometry under non-reducing conditions to characterize the nature of the LMW species.

[0180] The mass of the enriched LMW fraction was determined by mass spectrometry under native, non-reducing conditions using RP-HPLC coupled to electrospray ionization time-of-flight mass spectrometry. Results from the analysis indicate that the recovered LMW fraction is enriched in covalently linked light chain dimers (LC-LC) compared to the unfractionated bulk material.

[0181] The results of the analysis described in this example show that SE-UHPLC under non-denaturing conditions showed that Aimovig was present primarily in its monomeric form, as expected for an intact antibody containing two heavy chains and two light chains (approximately 99.2% of the drug substance). The remainder consisted of HMW species (0.5% of the drug substance) primarily composed of dimers and trace levels of LMW species. No higher-order oligomers were observed. SE-UHPLC fractions of Aimovig drug substance enriched in HMW species exhibited reduced inhibitory potency compared to the monomeric form of Aimovig.

[0182] The erenumab dimer is primarily composed of covalently linked monomeric subunits and does not dissociate under denaturing conditions. The majority of the dimer can be reduced and converted to its heavy and light chain components under reducing and denaturing conditions, which are therefore linked by disulfide bonds. The non-reducible species exists as a minor component of the covalently linked dimer and migrates after the heavy chain peak by rCE-SDS. Characterization of the SE-UHPLC LMW peak indicated that the light chain dimer is the enriched peak.

[0183] Several lots of commercial-scale manufactured erenumab drug substance (140 mg / mL) were analyzed by SE-UHPLC to assess the presence and amount of size variants (e.g., HMW and LMW species), as measured by the peak area percentage of the HMW and LMW peaks in the SE-UHPLC chromatogram. The potency of the drug substance lots was also evaluated by cell-based potency assays and compared to the potency of lot number 78137, the erenumab drug substance sample employed in Phase II / Phase III clinical trials. A summary of the data is provided in Table 27 below.

[0184] [Table 27]

[0185] As shown by the data in Table 27, commercial-scale manufactured erenumab drug substance contained a consistent level of approximately 0.6% HMW species as measured by SE-UHPLC. Drug substance containing this level of HMW species exhibited potency comparable to that of the erenumab drug substance employed in clinical trials.

[0186] Three of the drug substance batches from Table 27 (63131, 63132, and 63133) were stored at 25°C for three months, and samples were subjected to SE-UHPLC analysis and potency testing to assess any impact on drug substance potency due to changes in the level of size variants over time. The results of this stability study are shown in Table 28.

[0187] [Table 28]

[0188] The drug substance containing a high level of HMW species, 1.6%, had efficacy comparable to that of the erenumab drug substance employed in clinical trials.

[0189] To evaluate the susceptibility of Aimovig to forming size variants under heat stress conditions, Aimovig drug substance was incubated at 50°C for 14 days. Samples were removed at various time points throughout the duration of the test and frozen. After the final time point, all samples were analyzed in parallel to minimize analytical variability. Samples were assessed by SE-UHPLC according to the method described above and the cell-based bioassay described in Example 1.

[0190] The native SE-UHPLC profiles of erenumab drug substance stressed at 50°C on days 0 and 14 are shown in Figure 20. The relative peak area % for the peaks shown in Figure 20 is presented in Table 29 along with the % relative potency of the samples as assessed by cell-based bioassay. HMW species were the dominant degradants, showing an increase over 14 days with a corresponding decrease in the main peak. The HMW species produced were primarily composed of higher-order aggregates (HMW species greater than dimers). An increase in LMW species was also observed on day 14. Heat-stressed samples of erenumab drug substance exhibited a significant decrease in potency. The decrease in potency was primarily due to an increase in HMW species and Asp 105 These were also increased in heat-stressed samples of erenumab drug substance due to an increase in isomerization variants (see Example 2).

[0191] [Table 29]

[0192] Further heat stress testing was performed on additional erenumab drug substance batches manufactured at commercial scale. Three of the drug substance batches from Table 27 (63131, 63132, and 63133) were stored at 40°C for up to 30 days, and samples were subjected to SE-UHPLC analysis and cell-based potency testing to further assess any impact on drug substance potency due to changes in size variant levels over time. The results of this heat stress testing are shown in Table 30.

[0193] [Table 30]

[0194] The results of this study show a general trend of decreased potency with increasing HMW and LMW species. In particular, levels of HMW species greater than about 2.5% in the drug substance were associated with decreased potency of the drug substance. It is noted that the 30-day sample from lot 63132 did not show a decrease in potency, likely due to variability in the cell-based bioassay.

[0195] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0196] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the claims appended hereto. The present invention provides, for example, the following items. (Item 1) 1. A composition comprising Aimovig and one or more Aimovig variants, wherein the one or more Aimovig variants include isomerized variants and desamidated variants, and the amount of the isomerized variants and desamidated variants in the composition is less than about 30%. (Item 2) 2. The composition of claim 1, wherein the amount of the isomerized and deamidated variants in the composition is less than about 15%. (Item 3) 2. The composition of claim 1, wherein the amount of the isomerized and deamidated variants in the composition is less than about 8%. (Item 4) 2. The composition of claim 1, wherein the amount of the isomerized variants and deamidated variants in the composition is less than about 4%. (Item 5) 2. The composition according to item 1, wherein the amount of the isomerized variant and the deamidated variant in the composition is about 1% to about 10%. (Item 6) 2. The composition according to item 1, wherein the amount of the isomerized variant and the deamidated variant in the composition is about 1% to about 4%. (Item 7) 7. The composition of any one of items 1 to 6, wherein the isomerization variant has an isoaspartic acid residue or succinimide at amino acid position 105 in one or both heavy chains of erenumab (SEQ ID NO: 1 or SEQ ID NO: 3). (Item 8) 8. The composition of any one of items 1 to 7, wherein the desamido variant has an asparagine residue at amino acid position 102 in one or both heavy chains of erenumab (SEQ ID NO: 1 or SEQ ID NO: 3) converted to an aspartic acid residue, a succinimide residue, or an isoaspartic acid residue. (Item 9) 9. The composition according to any one of items 1 to 8, wherein the amount of the isomerization variant and the deamidation variant in the composition is determined by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC). (Item 10) A composition comprising Aimovig and one or more Aimovig variants, wherein the amount of Aimovig in the composition is less than about 40%. (Item 11) 11. The composition of claim 10, wherein the amount of acidic variant in the composition is less than about 35%. (Item 12) 11. The composition according to item 10, wherein the amount of the acidic variant in the composition is about 25% to about 38%. (Item 13) 11. The composition according to item 10, wherein the amount of the acidic variant in the composition is about 26% to about 34%. (Item 14) 14. The composition of any one of items 10 to 13, wherein the one or more acidic variants comprise disulfide isoform variants, fragmentation variants, or a combination thereof. (Item 15) The composition according to any one of Items 10 to 14, wherein the amount of the acidic variant in the composition is determined by cation exchange high performance liquid chromatography (CEX-HPLC). A composition comprising Aimovig and one or more disulfide isoform variants thereof, wherein the one or more disulfide isoform variants comprise an IgG2-B isoform, and the amount of the IgG2-B isoform in the composition is less than about 20%. (Item 17) 17. The composition of item 16, wherein the amount of the IgG2-B isoform in the composition is less than about 10%. (Item 18) Item 17. The composition according to item 16, wherein the amount of the IgG2-B isoform in the composition is about 4% to about 6%. (Item 19) 19. The composition of any one of items 16 to 18, wherein the one or more disulfide isoform variants further comprise an IgG2-A / B isoform. (Item 20) 20. The composition according to item 19, wherein the amount of the IgG2-A / B isoform in the composition is about 34% to about 37%. (Item 21) 21. The composition according to any one of items 16 to 20, wherein the amount of disulfide isoform variants in the composition is determined by non-reducing reverse-phase high performance liquid chromatography (RP-HPLC). (Item 22) 1. A composition comprising Aimovig and high molecular weight (HMW) species of Aimovig, wherein the amount of the HMW species in the composition is less than about 2.5%. (Item 23) 23. The composition of claim 22, wherein the amount of HMW species in the composition is about 1.8% or less. (Item 24) 23. The composition of claim 22, wherein the amount of HMW species in the composition is about 1.2% or less. (Item 25) 25. The composition of any one of items 22 to 24, wherein the HMW species comprises a covalently linked dimer of Aimovig. (Item 26) 26. The composition of any one of items 22 to 25, wherein the amount of HMW species in the composition is determined by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). (Item 27) 1. A composition comprising Aimovig and one or more Aimovig variants, wherein the Aimovig variants include isomerized variants, desamidated variants, acidic variants, HMW species, or a combination thereof, and wherein the composition has the following characteristics: (a) the amount of acidic variant in the composition is about 25% to about 38% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 2.1% or less, as measured by SE-UHPLC; and (c) the amount of isomerized and deamidated variants in the composition is about 8% or less, as measured by pre-peak in HIC-HPLC; A composition having one or more of: (Item 28) The composition has the following characteristics: (a) the amount of acidic variant in the composition is about 26.5% to about 33.6% as measured by CEX-HPLC; (b) the amount of HMW species in the composition is about 1.2% or less, as measured by SE-UHPLC; and (c) the amount of isomerized and deamidated variants in the composition is about 3.2% or less, as measured by pre-peak in HIC-HPLC; 28. The composition according to item 27, having one or more of: (Item 29) 29. The composition of any one of items 1 to 28, wherein erenumab comprises a heavy chain of SEQ ID NO: 1 and a light chain of SEQ ID NO: 2. (Item 30) 29. The composition of any one of items 1 to 28, wherein erenumab comprises a heavy chain of SEQ ID NO: 3 and a light chain of SEQ ID NO: 4. (Item 31) 31. A pharmaceutical formulation comprising the composition according to any one of items 1 to 30 and one or more pharmaceutically acceptable excipients. (Item 32) 32. A method for treating, preventing or reducing the occurrence of headache in a patient in need thereof, comprising administering to said patient a pharmaceutical formulation according to item 31. (Item 33) 33. The method of claim 32, wherein the headache is a migraine. (Item 34) 34. The method of claim 33, wherein the migraine is episodic migraine or chronic migraine. (Item 35) 31. The erenumab composition according to any one of items 1 to 30, for use in a method for treating, preventing, or reducing the incidence of headache in a patient in need thereof. (Item 36) 36. The erenumab composition according to item 35, wherein the headache is a migraine. (Item 37) 37. The erenumab composition according to item 36, wherein the migraine is episodic migraine or chronic migraine. (Item 38) 31. Use of the erenumab composition of any one of items 1 to 30 in the manufacture of a medicament for treating, preventing, or reducing the occurrence of headache in a patient in need thereof. (Item 39) 39. The use according to item 38, wherein the headache is a migraine. (Item 40) 40. The use according to item 39, wherein the migraine is episodic migraine or chronic migraine. (Item 41) 1. A method for assessing the quality of an erenumab composition, comprising: Obtaining an erenumab composition containing erenumab and one or more erenumab variants; measuring the amount of one or more Aimovig variants in the composition, wherein the Aimovig variants comprise an isomerized variant, a desamidated variant, an acidic variant, a HMW species, or a combination thereof; comparing the measured amount of the one or more Aimovig variants with a pre-defined reference standard; and If the comparison indicates that the predetermined reference standard is met, preparing a pharmaceutical formulation or pharmaceutical product of the erenumab composition. A method comprising: (Item 42) 42. The method of claim 41, wherein the amount of isomerized variants and deamidated variants is measured, and the predetermined reference standard is about 10% or less. (Item 43) Item 43. The method of item 42, wherein the amount of isomerized variants and desamidated variants in the erenumab composition is measured by HIC-HPLC. (Item 44) 42. The method of claim 41, wherein the amount of an acidic variant is measured and the predetermined reference standard is about 38% or less. (Item 45) Item 45. The method of item 44, wherein the amount of acidic variant in the erenumab composition is measured by CEX-HPLC. (Item 46) 42. The method of claim 41, wherein the amount of HMW species is measured and the predetermined reference standard is about 2.5% or less. (Item 47) 47. The method according to item 46, wherein the amount of HMW species is measured by SE-UHPLC. (Item 48) 48. The method of any one of items 41 to 47, wherein the erenumab composition is obtained from a Chinese hamster ovary (CHO) cell line expressing a nucleic acid encoding a heavy chain of SEQ ID NO: 1 and a nucleic acid encoding a light chain of SEQ ID NO: 2.

Claims

[Claim 1] The invention as described in the drawings.

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