Method for purifying dulaglycopeptides using hydrophobic interaction chromatography

Abstract

The present disclosure relates to methods of purifying dulaglycopeptide compositions. For example, the method includes in-line loading a dulaglycopeptide composition along with a buffer B solution in a downflow direction onto a hydrophobic interaction chromatography (HIC) column, washing the HIC column with a buffer mixture of a buffer A solution and a buffer B solution in the downflow direction, and eluting the dulaglycopeptide from the HIC column. The linear gradient of the percentages of the buffer solution A and the buffer solution B is used to elute dulatide, and then isocratic maintenance is performed based on the reading of the UV absorbance of the eluent. The method also includes maintaining isocratic retention until anterior cleavage is performed, and then lowering the buffer solution B solution for elution of dulatide from the HIC column.

Description

[0001] The preparation of recombinant Fc-containing proteins (e.g., dulaglutide) for therapeutic use typically involves expressing proteins in mammalian cells and subsequently purifying these proteins from host cell contaminants. Host cell contaminants include components of non-proteinaceous properties (e.g., DNA, RNA, lipids, etc.) and proteinaceous properties (e.g., peptides and proteins from host cells are generally referred to as host cell proteins (HCPs)). If these contaminants are not removed, they can retain their endogenous properties and degradation products (e.g., endogenous proteases) or affect patients treated with recombinant Fc-containing protein products. In addition, the durability of the purification elements (e.g., chromatographic resins) may be important in the cost and / or efficiency of the entire process. For example, selecting a resin that can be cleaned and reused several more times can reduce the frequency of resin replacement and can reduce the overall cost of a method that undergoes multiple runs. In addition, the method using the chromatographic resin can be optimized to improve efficiency.

[0002] Therefore, there is a need for improved and / or alternative methods for selecting and using chromatographic techniques and resins to effectively purify Fc-containing proteins to remove host cell contaminants.

[0003] Dulaglutide (LY2189265, GLP-Fc) is a glucagon-like peptide-1 (GLP-1) analog that has been fused to a modified Fc portion of human immunoglobulin G4 (IgG4) via a short, flexible peptide linker. It is the active ingredient in Trulicity®, which has been approved as an adjunct to diet and exercise to improve glycemic control in patients with type 2 diabetes. The dulaglutide molecule is used as a stand-alone therapy or in combination with orally active agents such as sulfonylureas (SUs), metformin (METs), thiazolidinediones (TZDs) to improve glycemic control in patients with type 2 diabetes (T2DM). Dulaglutide was initially approved by the FDA in 2014 and was approved in February 2020 for the prevention of cardiovascular events in patients with T2DM and multiple cardiovascular risk factors. It is the first T2DM drug approved to reduce the risk of major adverse cardiovascular events (MACE) in primary and secondary prevention populations. The structure, function, preparation, and use of dulaglutide in treating T2DM are described in more detail in U.S. Patent No. 7,452,966 and U.S. Patent Application Publication No. US 2010 / 0196405.

[0004] To produce dulaglutide, mammalian cell culture techniques are used to synthesize and secrete the dulaglutide protein. For example, the method involves expanding a working cell bank of dulaglutide, such as Chinese hamster ovary (CHO) cells, through a series of shake flasks and seed bioreactors to generate sufficient biomass for inoculating a production bioreactor. In the production bioreactor, dulaglutide is secreted from CHO cells cultured using several culture method steps to optimize, for example, product quality and process performance.

[0005] Once the cell culture meets defined criteria (e.g., culture time limit, titer / cell density, number of passages, number of doublings, and / or genetic stability), a primary recovery process for dulaglutide is performed from the production process. One example of a primary recovery process includes centrifugation to remove CHO cells and large, insoluble cell debris, followed by a normal flow filtration procedure to remove fine suspended particles. The resulting filtrate from the primary recovery process is then subjected to detergent viral inactivation (DVI) by adding detergent to the clarified filtrate. Following detergent inactivation, dulaglutide is purified from proteinaceous and non-proteinaceous components of the product stream by a protein A affinity capture chromatography step. The main stream of dulaglutide captured from the column is then subjected to low pH viral inactivation / neutralization, followed by protease heat inactivation. Following two corresponding inactivation steps, dulaglutide and product-related impurities, including host cell proteins (HCPs), are separated using anion exchange chromatography (AEX).

[0006] During several purification process steps, dulaglutide is measured, quantified, and identified as an IgG-GLP fusion protein. For example, during the affinity capture step, dulaglutide can be measured based on the IgG-GLP fusion protein content. For other IgG-specific quantitative assays, as well as the inherent specificity of downstream purity assays, dulaglutide can be identified by measuring the IgG-GLP fusion protein content. The amount of dulaglutide can also be monitored. For example, prior to affinity capture, dulaglutide protein content is monitored by using protein A affinity capture high performance liquid chromatography (HPLC). After the affinity capture chromatography step, protein purity is typically expected to be greater than 95% dulaglutide-related substances, and dulaglutide downstream processes can employ online spectrophotometric measurements.

[0007] The dulaglutide preparation process may generate one or more impurities, such as host cell proteins (HCP), host cell DNA, residual protein A (rProA), residual Triton X-100, and residual insulin. Acceptable reduction and / or removal of HCP, DNA, and rProA can be monitored and measured at various steps in the purification process. Monitoring the reduction and / or removal of these impurities in the dulaglutide drug substance preparation process can also confirm process performance and consistency. Remaining process-related impurities (e.g., residual Triton X-100, residual insulin) can also be tested and measured.

[0008] Additional purification steps are used to further purify dulaglutide and reduce one or more impurities from the production process, for example, after the AEX described above. For example, a ceramic hydroxyapatite (CHT) column can be used for additional chromatographic purification. However, the lifespan of CHT columns used in the production process is limited, they can be expensive, and they can cause downtime when the CHT resin needs to be replaced. Therefore, the applicant seeks to develop additional chromatographic purification methods to, for example, extend the lifespan of the resin and / or maintain the functional aspects of purification, i.e., to increase the purity of dulaglutide and remove impurities from the dulaglutide production process.

[0009] Overview

[0010] The present invention generally relates to a method for purifying a dulaglutide composition using a hydrophobic interaction chromatography (HIC) column. Such a method comprises a loading step, a washing step, and an elution step. The loading step comprises loading the dulaglutide composition and a buffer B solution onto the HIC column in a downstream direction. The washing step comprises washing the HIC column with a buffer mixture of buffer A and buffer B solutions. The elution step comprises eluting the dulaglutide from the HIC column using a linear gradient of percentages of buffer A and buffer B solutions, followed by an isocratic hold based on UV absorbance readings of the eluate from the HIC column, indicating that protein-containing material is eluting from the HIC column. The method further comprises maintaining the isocratic hold and then reducing the buffer B solution to 0% to elute the dulaglutide from the HIC column.

[0011] In some embodiments, a method for purifying a dulaglutide composition using a hydrophobic interaction chromatography (HIC) column comprises loading the dulaglutide composition and a buffer B solution onto the HIC column in a downstream direction at a volume ratio ranging from 62.7% to 72.5% and a linear velocity ranging from 80 cm / hr to 160 cm / hr, wherein dulaglutide from the dulaglutide composition is bound to the HIC column, and the composition comprises a plurality of compounds from a mammalian cell fermentation process. The method further comprises washing the HIC column with a buffer mixture comprising 15% to 25% of a buffer A solution and 75% to 85% of a buffer B solution in a downstream direction at a linear velocity ranging from 80 cm / hr to 160 cm / hr for a period of 2.7 column volumes (CV) to 3.3 CV. The method then involves eluting dulaglutide from the HIC column using a linear gradient comprising 67.5% buffer B solution to 10% buffer B solution for a period of 10 CV to 12 CV, followed by an isocratic hold based on UV absorbance readings of the eluate at A 280 nm in the range of 0.1 AU / cm to 0.5 AU / cm, indicating that the protein-containing material has eluted from the HIC column. The isocratic hold was maintained for 1.25 CV to 1.75 CV, at which time a frontal cut was performed and mainstream collection began as the elution gradient decreased the buffer B solution from its isocratic hold to 0%.

[0012] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Exemplary embodiments are illustrated in the accompanying drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0016] Figure 1 The unit operations for purifying a dulaglutide composition using a HIC column according to some embodiments herein are illustrated.

[0017] Figure 2 1 is a chromatogram overlay of one embodiment of the present disclosure for purifying a dulaglutide composition.

[0018] Figure 3 1 is a comparative chromatographic overlay of four types of HIC column resins involved in the methods according to some embodiments described herein.

[0019] describe

[0020] Before describing the present compositions and methods, it should be understood that the present disclosure is not limited to the described compositions, methods, or regimens comprising dulaglutide, as these may vary. It should also be understood that the terminology used in this specification is for the purpose of describing particular forms or embodiments only and is not intended to limit the scope of the present disclosure, which will be limited solely by the appended claims.

[0021] The terms used herein have meanings that are recognized and known to those skilled in the art, however, for convenience and completeness, specific terms and their meanings are set forth below.

[0022] In addition, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "sphere" refers to one or more spheres and their equivalents known to those skilled in the art, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure, preferred methods, apparatus, and materials are described below. All publications cited herein are incorporated herein by reference. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure with respect to prior disclosure.

[0023] As used herein, the term "about" when used in conjunction with a specific value is meant to include acceptable deviations from that value. In certain embodiments, the term "about" is meant to include values ​​that are 1%, 3%, 5% or 10% higher or lower than a given value.

[0024] As used herein, the term "dulaglutide" refers to any GLP-1 receptor agonist protein dimer of two monomers having the amino acid sequence of SEQ ID NO: 1, including any protein that is the subject of a regulatory submission seeking approval of a GLP-1 receptor agonist product that relies, in whole or in part, on data related to dulaglutide submitted to regulatory agencies by Eli Lilly and Company, regardless of whether the party seeking approval of the protein actually identifies the protein as dulaglutide or uses another term. Dulaglutide agonizes the GLP-1 receptor, thereby stimulating insulin synthesis and secretion, and has been shown to provide improved glycemic control in patients with T2DM. Each monomer of dulaglutide has the amino acid sequence set forth in SEQ ID NO: 1.

[0025] (SEQ ID NO: 1)

[0026] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0027] As used herein, the term "mammalian cell" refers to cells from any mammalian species, including but not limited to mouse, rat, rabbit, dog, primate, and, for example, human.

[0028] As used herein, hydrophobic interaction chromatography (HIC) is a method for separating proteins based on the difference in protein surface hydrophobicity by utilizing the reversible interaction between the protein and the HIC resin hydrophobic surface. Typically, the binding between the hydrophobic protein and the HIC resin ligand is affected by the presence of ionic interactions of the running buffer and / or mobile phase. High salt concentrations can enhance this interaction, while reducing salt concentrations can weaken this interaction. As the ionic strength of the buffer solution decreases, the interaction between the protein and the matrix / resin is reversed, and first elution has the lowest degree of hydrophobicity. The most hydrophobic protein is eluted last, thereby needing to reduce salt concentration to a greater extent to reverse this interaction.

[0029] As used herein, the term "isocratic hold" refers to the period during which the ratios of the individual buffer solutions in a mixture of two buffer solutions remain constant over the entire chromatographic test time.

[0030] As used herein, the term "buffer solution" may alternatively be defined as "mobile phase" or "solvent" or "eluent" or "medium." The term "buffer" refers to a solution that maintains the pH of a solution within a certain range and increases the amount of acid or base that must be added to cause a unit change in pH.

[0031] As used herein, the term "downflow direction" can refer to flow in the opposite or opposite direction to the "upflow direction", or vice versa. Separation can be achieved by pumping the buffer solution or mobile phase upward or against gravity through the HIC column, or by causing the buffer solution or mobile phase to flow downward or toward gravity through the HIC column. Those skilled in the art will understand that the column can be operated in a "downflow" mode as described above, or in an "upflow" mode in which the flow direction of the mobile phase is reversed, causing it to move upward through the HIC column. In the downflow mode, the buffer solution or mobile phase can enter the HIC column through the top, move vertically along the column bed, and flow downward or toward gravity through the column bed, and ultimately exit the HIC column through the bottom. Conversely, in the upflow mode, the buffer solution or mobile phase can enter the HIC column through the bottom, move vertically along the column bed, and flow upward or against gravity through the column bed, and ultimately exit the HIC column through the top.

[0032] As used herein, the term "eluent" refers to a liquid medium containing a molecule of interest, such as dulaglutide or a protein-containing material, which can be obtained after the molecule of interest is bound to a HIC column and an elution buffer solution is added to dissociate the molecule of interest from the HIC column.

[0033] As used herein, the term "linear gradient" refers to a technique in which the composition of the mobile phase or buffer solution is varied during a chromatographic run such that the composition of the buffer mixture changes linearly with time.

[0034] As used herein, the term "loading" or alternatively "charge" refers to the amount of molecule of interest (e.g., dulaglutide or protein-containing material) introduced into a HIC column, expressed as grams of molecule of interest per liter of bound material (e.g., resin) in the HIC column.

[0035] As used herein, the term "washing" refers to the process of releasing unbound molecules to the HIC column, such as impurities present in the loading solution, so that they flow with the solvent or mobile phase used.

[0036] The present disclosure relates to a method for purifying a dulaglutide composition, the method comprising: loading the dulaglutide composition onto a hydrophobic interaction chromatography (HIC) column online along with a buffer B solution in a downstream direction, wherein dulaglutide from the dulaglutide composition binds to the HIC column; washing the HIC column with a buffer mixture of buffer A and buffer B solutions; and eluting the dulaglutide from the HIC column using a linear gradient of percentages of buffer A and buffer B solutions, wherein the linear gradient is followed by an isocratic hold based on a UV absorbance reading of an eluate from the HIC column, the isocratic hold being maintained until front cleavage is performed, and then the buffer B solution is reduced to 0% for eluting the dulaglutide from the HIC column.

[0037] load

[0038] As disclosed herein, the method of the present disclosure comprises loading a dulaglutide composition together with a buffer B solution onto a hydrophobic interaction chromatography (HIC) column in a downstream direction, wherein dulaglutide from the dulaglutide composition binds to the HIC column.

[0039] In some embodiments, when the dulaglutide composition is loaded online onto the HIC column together with the buffer B solution, the ratio between the dulaglutide composition and the buffer B solution can range from 37.5%:62.7% to 27.5%:72.5% of the dulaglutide composition:buffer B solution.

[0040] In some embodiments, loading the Buffer B solution onto the HIC column together with the dulaglutide composition can increase the hydrophobicity of the dulaglutide in the composition and ensure that the dulaglutide binds to the HIC column resin.

[0041] In some embodiments, when the dulaglutide composition is loaded online onto a HIC column along with a buffer B solution, the target loading capacity can range from 10 g to 20 g of dulaglutide per liter of HIC resin.

[0042] In some embodiments, loading the dulaglutide composition onto the HIC column can be performed at a linear flow rate of 80 centimeters per hour (cm / hr) to 160 cm / hr. In some embodiments, the linear velocity can be 120 cm / hr.

[0043] In some embodiments, the dulaglutide composition can be a downstream product of an anion exchange chromatography column from a dulaglutide preparation and purification process. The dulaglutide composition can contain a variety of compounds or impurities, including host cell proteins (HCP), residual host cell proteins (rHCP), host cell DNA (HCDNA), residual protein A (rProA), residual Triton X-100, and residual insulin.

[0044] In some embodiments, the concentration of the protein-containing material in the dulaglutide composition may not exceed 7.8 grams per liter (g / L). In some embodiments, the concentration of HCPs in the dulaglutide composition loaded onto the HIC column may range from 30 ppm to 70 ppm. Most of the HCP removal may occur before the anion exchange chromatography unit, and the HCP concentration may then decrease during the anion exchange chromatography unit operation. The HCPs may then be removed in other unit operations before the anion exchange chromatography unit (e.g., affinity capture chromatography unit and low pH viral inactivation unit). Therefore, the concentration of HCPs in the dulaglutide composition loaded onto the HIC column may be negligible compared to the concentration of dulaglutide in the loaded composition.

[0045] In some embodiments, the concentrations of host cell DNA (HCDNA), residual protein A (rProA), residual Triton X-100, and residual insulin in the dulaglutide composition loaded onto the HIC column may be negligible compared to the concentration of dulaglutide. Most of the impurities—HCDNA, rProA, residual Triton X-100, and residual insulin—can be removed in a unit operation prior to the anion exchange chromatography unit, followed by removal of HCDNA, rProA, residual Triton X-100, and residual insulin in an anion exchange chromatography unit operation prior to the HIC unit. In some embodiments, trace amounts of these impurities may be present in the dulaglutide composition.

[0046] Buffer B solution

[0047] In some embodiments, the buffer B solution may comprise a salt selected from the group consisting of ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium bromide, and combinations thereof. In some embodiments, the buffer B solution comprises sulfate, tris(hydroxymethyl)aminomethane (Tris), or combinations thereof. The sulfate may be selected from the group consisting of ammonium sulfate and sodium sulfate. In some embodiments, the buffer B solution may comprise a salt selected from the group consisting of Ba 2+ , Ca 2+ Mg 2+ 、Na + , K + , Rb + and NH4 + cations, and / or selected from PO4 3- 、SO4 2- 、CH2CO3、Cl - Br - 、NO3 - , I - 、SCN - or anions of combinations thereof.

[0048] In some embodiments, the buffer B solution comprises Tris and sodium sulfate salts. For example, a dulaglutide composition loaded online with the buffer B solution may comprise 20 millimolar (mM) Tris and 400 mM sodium sulfate in the buffer B solution.

[0049] In some embodiments, the conductivity of the buffer B solution can be maintained by maintaining the concentration of the buffer B solution within a target range for the HIC unit operation. For example, a buffer B solution concentration range of 328.9 mM to 455.2 mM can produce a conductivity that is ± 6 millisiemens / centimeter (mS / cm) different from the target conductivity range of the buffer B solution.

[0050] HIC Column

[0051] As disclosed herein, the method includes loading a dulaglutide composition online with a buffer B solution onto a HIC column. Hydrophobic interaction chromatography (HIC) is a purification technique based on the interaction of materials, such as the resin in the HIC column, with the hydrophobic regions of molecules. Depending on the partition coefficient of the molecule in the medium in contact with the HIC column, the HIC column can preferentially bind a molecule of interest from a mixture of molecules in the medium. As disclosed in the methods herein, after loading the dulaglutide composition online with a buffer B solution, the dulaglutide can bind to the HIC column resin.

[0052] In some embodiments, the HIC column can have a diameter in the range of 50 cm to 200 cm. For example, a 200 cm diameter HIC column can be used in the HIC unit operation.

[0053] In some embodiments, the HIC column can have a packed bed height in the range of 15 cm to 30 cm. For example, the HIC column can have a packed bed height of 21 cm.

[0054] In some embodiments, the HIC column can comprise a material with high mechanical strength that can withstand high pressures. For example, the HIC column can comprise a stainless steel body.

[0055] In some embodiments, the HIC column comprises one or more upstream in-line filters. For example, the HIC column can comprise two 0.2 micron (μm) filters upstream of the column. For example, the HIC column can comprise a 0.45 / 0.22 μm size polyvinylidene fluoride or PVDF filter (30 inch column), which can then be referred to as the loading or charging filter, followed by an additional 0.5 / 0.2 μm polyethersulfone or PES filter (30" column), which can be referred to as the column pre-filter. The loading or charging filter can be in-line only for equilibration, loading or charging, and column washing. The pre-filter can be in-line whenever the column is in-line except for loading or charging, column cleaning and sanitizing.

[0056] In some embodiments, the HIC column can include a resin having at least one hydrophobic ligand. For example, the resin used in the HIC column can be selected from alkyl-, aryl-, and combinations thereof. For example, the HIC medium can be selected from butyl, hexyl, phenyl, octyl, and polypropylene glycol ligands. In some embodiments, the HIC column can include Capto phenyl as a hydrophobic ligand.

[0057] In some embodiments, the HIC unit operation can be performed using Capto-Phenyl ImpRes resin, which is typically packed to a bed height of 15-30 cm. The target bed height for the HIC column can be 21 cm. In some embodiments, the HIC column can have a bed height of 10 cm.

[0058] In some embodiments, the HIC column can have a column volume of 4.7 mL.In some embodiments, the HIC column resin can have a hydrophobicity that results in 45-50 min lysozyme retention.

[0059] In some embodiments, the HIC column resin can have a flow rate of up to 220 cm / h at 20°C in a 1 m diameter column with a 20 cm bed height, which can be measured using the same method buffer as the viscosity of water at 300 kPa.

[0060] In some embodiments, the HIC column resin can have a binding capacity of 19 mg bovine serum albumin (BSA) per mL of solvent or medium.

[0061] In some embodiments, the HIC column resin can have a pH stability of pH 2 to 14. For example, the HIC resin can be stable in 1 M NaOH.

[0062] In some embodiments, HIC column resin can be stored in 20% ethanol at 4°C-30°C.

[0063] In some embodiments, the HIC column can comprise a highly cross-linked agarose matrix having phenyl ligands. In some embodiments, the HIC column can comprise a matrix having an average particle size of 40 μm.

[0064] In some embodiments, the HIC column matrix can have a pH stability of pH 2-14.

[0065] In some embodiments, a target flow rate of 120 cm / hr may be applied during the online loading of the dulaglutide composition with the buffer B solution onto the HIC column.

[0066] washing

[0067] As disclosed herein, the methods of the present disclosure include washing the HIC column with a buffer mixture of a buffer A solution and a buffer B solution. In some embodiments, buffer A can be Tris or tris(hydroxymethyl)aminomethane. For example, buffer A can contain 20 mM Tris. The conjugate acid of Tris has a partition coefficient (pKa) of 8.07 at 25°C, which means that buffer A can have an effective pH range of 7.1-9.1 (pKa ± 1) at room temperature.

[0068] The buffer mixture can include the same source of buffer B solution as the online buffer B solution used in the loading step. In some embodiments, the buffer mixture can include a buffer B solution from a different source than the buffer B solution used in the loading step, but having the same concentration. As disclosed, buffer B can include Tris and sodium sulfate salts. For example, buffer B can include 20 mM Tris and 400 mM sodium sulfate for washing the HIC column.

[0069] The buffer mixture may comprise a salt selected from the group consisting of ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium bromide, and combinations thereof. In some embodiments, the buffer mixture may comprise sulfate, tris(hydroxymethyl)aminomethane, or a combination thereof. The sulfate may be selected from the group consisting of ammonium sulfate and sodium sulfate. In various embodiments, the buffer mixture may comprise a salt selected from the group consisting of Ba 2+ , Ca 2+ Mg 2+ 、Na + , K + , Rb + and NH4 + cations, and / or selected from PO4 3- 、SO4 2- 、CH2CO3、Cl - Br - 、NO3 - , I - 、SCN - and combinations thereof.

[0070] In some embodiments, the buffer A solution can comprise 20 mM Tris at a pH range of 7.7-8.3, and the buffer B solution can comprise 20 mM Tris, 400 mM sodium sulfate at a pH range of 7.7-8.3. Thus, the buffer mixture can comprise a 20 mM Tris, 320 mM sodium sulfate solution at a pH range of 7.7-8.3.

[0071] In some embodiments, the pH of the buffer mixture can be controlled throughout the process as a function of the buffer composition and process step requirements and can be monitored as an overall aspect of the unit operation rather than as a test within a specific discrete process. pH control for viral inactivation can be controlled by performing an online pH test in the manufacturing facility prior to the anion exchange chromatography unit operation.

[0072] In some embodiments, the buffer mixture used to wash the HIC column can contain 15%-25% buffer A solution and 75%-85% buffer B solution. For example, the buffer mixture can contain 20% buffer A solution and 80% buffer B solution for washing the HIC column.

[0073] In some embodiments, washing the HIC column can include applying the buffer mixture at a linear velocity of 80 cm / hr to 160 cm / hr. In some embodiments, the linear velocity can be 120 cm / hr.

[0074] In some embodiments, washing the HIC column can include using 2.7 column volumes (CV) to 3.3 CV of the buffer mixture. For example, washing the HIC column can include using 3.0 CV of the buffer mixture.

[0075] Elution

[0076] As disclosed herein, the method of the present disclosure comprises eluting dulaglutide from a HIC column using a linear gradient of percentages of buffer A and buffer B solutions followed by an isocratic hold. The isocratic hold can be based on UV absorbance readings of the HIC column eluate. The method further comprises maintaining the isocratic hold until frontal cleavage is performed, and then reducing the percentage of buffer B solution to 0% for elution of dulaglutide from the HIC column.

[0077] Loading the dulaglutide composition onto the HIC column online along with the buffer B solution results in dulaglutide binding to the HIC column. For example, dulaglutide can be eluted from the HIC column when the salt concentration in the buffer mixture is reduced linearly or stepwise. In some embodiments, when the HIC column is washed with the buffer solution, dulaglutide may remain bound to the HIC column, while one or more impurities present in the composition may be washed with the buffer solution. When the elution phase is initiated, dulaglutide may begin to elute from the HIC column.

[0078] As disclosed herein, elution of dulaglutide from the HIC column can be performed by a linear gradient step of the percentages of buffer A and buffer B solutions, followed by an isocratic hold step, and by decreasing the percentage of buffer B solution to 0%. For example, the linear gradient decreases the percentage of buffer B solution while increasing the percentage of buffer A solution. In some embodiments, the percentages of buffer A and B solutions and the timing of the gradient are automated, for example, based on several factors, such as column volume, UV signal, etc.

[0079] In some embodiments, eluting dulaglutide from the HIC column can include using a buffer B solution with a linear gradient ranging from 67.5% to 72.5% of the buffer B solution at a selected starting point and a linear gradient ranging from 5% to 15% of the buffer B solution at a selected ending point.

[0080] In some embodiments, eluting dulaglutide from the HIC column can include using a buffer A solution with a linear gradient ranging from 32.5% to 27.5% of the buffer A solution at a selected starting point and a linear gradient ranging from 95% to 85% of the buffer A solution at a selected ending point.

[0081] In some embodiments, the duration of the linear gradient duration varies based on the composition of the buffer A and buffer B solutions. In some embodiments, eluting dulaglutide with a linear gradient comprises 10 column volumes (CV) to 12 CV of buffer A and buffer B solutions. For example, eluting dulaglutide with a linear gradient can comprise 11.5 CV of buffer A and buffer B solutions. In some embodiments, a linear gradient from 67.5% buffer B solution (320 mM sodium sulfate) to 10% buffer B solution (40 mM sodium sulfate) can be completed over 11.5 CV.

[0082] In some embodiments, eluting dulaglutide with a linear gradient can include a linear gradient of 80 cm / hr to 160 cm / hr of a buffer mixture comprising a solution of buffer A and buffer B. In some embodiments, the linear flow rate of the buffer mixture for eluting dulaglutide from the HIC column can be 120 cm / hr.

[0083] As disclosed herein, elution of dulaglutide from the HIC column also includes an isocratic hold step. In some embodiments, the isocratic hold can be based on UV absorbance readings of the eluate from the HIC column. In some embodiments, the eluate can be a material containing protein, such as one or more impurities and / or dulaglutide, depending on, for example, the percentages of Buffer A and Buffer B solutions, UV absorbance readings, etc. UV absorbance readings can be measured at a wavelength of 280 nanometers (nm).

[0084] In some embodiments, isocratic maintenance can be carried out with 10% buffer B solution.For example, isocratic maintenance can be carried out with 10% buffer B solution of 2.0 CV.The column volume of buffer B solution used can indicate the duration of isocratic maintenance.If front side cutting does not occur in the elution gradient, it can be built into automation.In the elution process, once the UV signal of 0.25 absorbance unit (AU / cm) per centimeter is reached, the linear gradient can be switched to isocratic conditions, to maintain the constant concentration of salt.For example, once the UV signal of 0.25 AU / cm is reached, the linear gradient can be switched to isocratic conditions, to maintain the constant concentration of sodium sulfate.

[0085] In some embodiments, the frontal cleavage can be performed after 1.5 CV of isocratic flow. The mainstream product collection step can then begin. Dulaglutide may not elute from the HIC column, or may remain bound to the HIC column resin during the wash step, and elution can begin when a linear gradient is applied or after a static hold at 0% buffer B.

[0086] In some embodiments, a buffer mixture may be used to remove or wash impurities from the dulaglutide composition prior to the elution step.

[0087] Impurities can be removed on the front side of the main stream, and thus, once the UV front cut target is reached, the elution gradient can be reduced to 0% buffer B to complete the elution. In some embodiments, the target A280 cut point on the back side of the main stream can be 0.25 AU / cm. After the main stream (i.e., dulaglutide) has eluted, the elution can be terminated early by continuing with the column regeneration step, for example, as described below.

[0088] In some embodiments, the dulaglutide composition purification method can increase the concentration of dulaglutide initially present in the loaded dulaglutide composition. For example, the dulaglutide composition purification method can increase the concentration of dulaglutide in the mainstream product to about 2.15 g / L to 6.51 g / L after elution from the HIC column.

[0089] In some embodiments, the dulaglutide composition purification method can also reduce, for example, trace impurities initially present in the dulaglutide composition load. For example, the dulaglutide composition purification method can further reduce the concentration of host cell protein in the final main stream of the dulaglutide composition from a range of 30-70 ppm to approximately 10 ppm. Furthermore, for example, the dulaglutide composition purification method can reduce the concentration of host cell protein in the main stream product after elution from the HIC column, for example, the host cell protein concentration after elution from the HIC column can range from 2 ppm to 11 ppm.

[0090] In some embodiments, dulaglutide in the main stream of the HIC product can be filtered through 0.2 μm as an element of bioburden control prior to commencing non-method hold time (NPHT) at 2-8°C.

[0091] Additional Steps

[0092] According to one or more embodiments, the disclosed methods for purifying dulaglutide compositions may include additional steps as needed. These steps may include balancing the column prior to HIC unit operation, regenerating the HIC column after use, sterilizing the HIC column before or after use, and storing the HIC column after use. One or more of the additional steps may be performed in the method for purifying the dulaglutide composition. Furthermore, in some embodiments, one or more of the additional steps may be performed in any combination.

[0093] balance

[0094] In some embodiments, the methods of the present disclosure further comprise equilibrating the HIC column in a downstream direction prior to loading the dulaglutide composition and the buffer B solution onto the HIC column in-line, wherein the linear gradient is 2 CV of 32.5% buffer A and 67.5% buffer B solution. For example, the HIC column can be equilibrated in a downstream direction using 32.5% 20 mM Tris, pH approximately 8.0 and 67.5% 20 mM Tris, 400 mM sodium sulfate, pH approximately 8.0. Equilibration of the HIC column is typically performed in a downstream direction.

[0095] regeneration

[0096] In some embodiments, the methods of the present disclosure further comprise regenerating or stripping the HIC column after elution is complete. For example, 3.0 CV of 0.01 N NaOH (diluted online from 0.1 N NaOH) can be applied to the HIC column in an upstream direction to regenerate the HIC resin. In some embodiments, a regeneration step can be performed after each elution step to ensure efficient HIC column operation.

[0097] disinfect

[0098] In some embodiments, the methods of the present disclosure further comprise sanitizing the HIC column. For example, sanitizing comprises washing the HIC column with 1.0 N NaOH in an upstream direction, wherein the target contact static hold time is no less than 30 minutes. For example, the HIC column can be sanitized with 2.0 CV of 1 N NaOH (diluted online from 3 N NaOH), followed by a target contact static hold time of no less than 30 minutes.

[0099] store

[0100] In some embodiments, the methods of the present disclosure also include two techniques for storing the HIC column after use, depending on the time the HIC column is maintained. For example, when the HIC column is maintained for more than 24 hours before loading the dulaglutide composition, storing the HIC column may include washing the HIC column with a storage buffer solution after use. In some embodiments, the storage buffer solution may be 0.01 N NaOH or its equivalent. For example, storing the HIC column may include washing the HIC column with 2.0 CV of 0.01 N NaOH storage buffer solution in an upstream direction. In some embodiments, storing the HIC column may be performed in an upstream direction for 2.0 CV. In some embodiments, storing the HIC column may include supplementing the HIC column with 100 mM NaCl in a 0.01 N NaOH column storage solution.

[0101] In other embodiments, the HIC column can be re-equilibrated when the HIC column is stored for less than 24 hours prior to loading the dulaglutide composition. For example, the equilibration step can be performed to store the HIC column for less than 24 hours and then perform another method of purifying the dulaglutide composition.

[0102] Example

[0103] The following examples are merely illustrative of the methods of the present disclosure and are not intended to limit the scope of the present disclosure, which is set forth in the claims.

[0104] Example 1

[0105] In Example 1, which relates to a dulaglutide purification unit comprising an HIC column of the present disclosure and a method for purifying a dulaglutide composition, example embodiments of the present disclosure are provided. For example, Figure 1 A dulaglutide purification unit is illustrated, comprising a holding vessel, a charging filter, a prefilter column, an HIC column, and online and offline processing for processing a dulaglutide composition. The dulaglutide purification unit also includes a flow controller, a pressure controller, an air sensor, a pH sensor, a temperature sensor, and a pressure sensor. The dulaglutide purification unit is also illustrated, along with methods performed according to the present disclosure.

[0106] exist Figure 1 In the embodiment of the present invention, a dulaglutide composition is loaded onto a dulaglutide purification unit from a downstream product of a chromatography process (e.g., an anion exchange chromatography unit). The composition is loaded into two holding vessels 102 and 104 on the dulaglutide purification unit via feed line 112. A first sensor 114 is aligned with an inline position of holding vessel 102. This configuration is used to measure, for example, the temperature, pressure, stirring speed, and volume of the composition loaded into holding vessel 102.

[0107] Similarly, a second sensor 116 is aligned in-line with holding vessel 104 and is configured to measure, for example, the temperature, pressure, agitation rate, and volume of the composition being charged to holding vessel 104. A combined stream 126 from holding vessels 102 and 104 is fed into charge filter 106. Two sets of sensors 118 and 120 are positioned in-line parallel with combined stream 126 and are configured to measure the flow rate and pressure of combined stream 126.

[0108] After filtering the dulaglutide composition in the loading filter 106, the dulaglutide composition is filtered using a pre-column filter 108 before being loaded onto the HIC column 110. Another set of sensors 122 is aligned with the in-line position where the dulaglutide composition is loaded. This configuration is used to measure the air, pH, temperature, and pressure of the dulaglutide composition as it is purified in the HIC column 110.

[0109] As disclosed, the HIC column is used to improve the overall purity of dulaglutide by increasing the purity of the reversed-phase main peak and reducing HCPs. UV spectroscopy unit 128 is connected to the HIC unit via connection line 130 and is configured to measure the absorbance of protein-containing material in the HIC product stream 124 at any given time. Based on the UV absorbance readings, the HIC column operating parameters are automatically modified to capture dulaglutide and release one or more impurities eluting from the HIC column.

[0110] Example 2

[0111] Examples of chromatograms generated during the dulaglutide purification unit and method described in Example 1 are described in Examples 2 and Figure 2 middle.

[0112] For example, Example 2 illustrates the overlay of chromatograms during the operation of an HIC column for purifying a dulaglutide composition. Figure 2 As shown in , chromatograms are generated during several steps of the disclosed method, including sanitization, equilibration, column loading, washing, elution and regeneration, disinfection and / or storage. Figure 2 Included is information regarding the UV-absorption of the dulaglutide composition as it is processed at different stages of the purification process using a dulaglutide purification procedure employing a HIC column. Figure 2 The chromatogram also includes data on the conductivity, pH, and % gradient of the buffer B solution.

[0113] Buffer B solution

[0114] like Figure 2As shown in , the HIC column is sanitized using approximately 2.0 column volumes (CV) of NaOH. Next, the HIC column is equilibrated with approximately 2.0 CV of a 67.5% buffer B solution, and then the column is loaded with the dulaglutide composition. The percentage of buffer B solution compared to buffer A solution can be maintained stable at approximately 67.5% B solution throughout the dulaglutide composition loading step.

[0115] After the disinfection and equilibration steps, the HIC column was loaded and then washed with a buffer mixture of Buffer A and Buffer B, i.e., approximately 3.0 CV of 80% Buffer B, to remove residual loading composition components. After the wash step, elution began with approximately 67.5% Buffer B. The percentage (%) of Buffer B decreased linearly from 67.5% until a UV signal of 0.25 AU / cm was reached. During this linear gradient elution step, the percentage of Buffer A increased as the percentage of Buffer B decreased. The linear gradient duration of the elution step utilized approximately 5 CV of Buffer A and Buffer B solutions.

[0116] During the linear gradient of the elution step, once a UV signal of 0.25 AU / cm was reached, the linear gradient was switched to an isocratic hold, maintaining a constant percentage of buffer B solution. The isocratic hold step was maintained at a constant percentage of approximately 40% buffer B solution and approximately 1.5 CV of each buffer A and buffer B solution was used, as shown in FIG. Figure 2 Then, the frontal cut is made and the main flow acquisition starts.

[0117] Impurities (e.g., protein-containing materials) present in the loaded dulaglutide composition are cleared on the front side of the main stream, and thus, once the UV front side cut target is reached, the elution gradient is reduced to 0% buffer B solution, thereby completing the elution of dulaglutide, as shown in FIG. Figure 2 . Mainstream collection of the eluate containing dulaglutide continued until a UV signal of 0.25 AU / cm was reached, at which point back-cutting was performed to complete elution. After complete elution, the final steps of HIC column regeneration, disinfection, and storage were performed. During these HIC column regeneration, disinfection, and storage steps, the percentage of Buffer B solution was maintained at 0%, as indicated.

[0118] pH and conductivity

[0119] like Figure 2As shown in , after the HIC column was sterilized and equilibrated before use, the pH of the assay solution remained stable at approximately 8.0 throughout the dulaglutide composition loading step, the washing step, and the elution step. When both the buffer A solution and the buffer B solution were at pH 8.0, changes in their concentrations at different stages of these operations did not cause any changes in the pH of the assay solution containing dulaglutide.

[0120] UV absorbance

[0121] The UV absorbance of the assay solution at A280 nm indicates the presence of dulaglutide and / or protein-containing material in the assay solution. As illustrated, the chromatogram also shows that the UV absorbance of the assay solution at A280 nm is near zero during all steps, including pre-use disinfection, equilibration, column loading, washing, and the first phase of the elution step before the second phase of the elution step. This confirms that dulaglutide or protein-containing material is bound to the HIC column, for example, during the loading step and / or remains bound to the HIC column during the washing and first phases of the elution step. Therefore, after 1.5 CV of isocratic flow, a frontal cut is performed and mainstream collection is initiated. Impurities or protein-containing material are cleared on the front side of the mainstream, and thus, once the frontal cut target is reached, the elution gradient is reduced to 0% Buffer B, completing the elution of dulaglutide. The target A280 cut point for the rear mainstream is 0.25 AU / cm.

[0122] At the end of the first phase of the elution step (referred to as the linear gradient phase of the elution step) and at the beginning of the second phase of the elution step (referred to as the isocratic hold phase of the elution step), the UV absorbance of the eluate at A 280 nm slowly increases. This indicates that material containing proteins, such as dulaglutide, is eluting from the HIC column.

[0123] The UV absorbance of the assay solution reaches a peak before rapidly decreasing to a level similar to that during the elution linear gradient step or any preceding step. This sharp peak in the UV absorbance reading at 280 nm also indicates the purity of the dulaglutide molecule or protein-containing material in the assay solution.

[0124] Example 3

[0125] Example 3 provides an exemplary selection of HIC column resins for use in the methods of the present disclosure. Four different types of HIC column resins were tested to determine their effectiveness in purifying dulaglutide compositions using the methods disclosed herein. The four types of HIC column resins were Phenyl Sepharose HP (PSP), Butyl Sepharose HP (BSP), Capto Phenyl Impres (CPI), and Toyopearl Phenyl 650S resin (TPP).

[0126] like Figure 3 As shown in , a comparative study between these four different HIC column resins showed that although each test run contained the same loading of dulaglutide composition to start the chromatography unit operation, the UV absorbance of the protein-containing material at a wavelength of A 280 nm was significantly different. The intensity and sharpness of the peak absorbance indicate the yield % of dulaglutide. Figure 3 As shown in , the performance of the resins follows the order: CPI>BSP>PSP>TPP. Table 1 below shows a summary of the % yields obtained using each of these HIC resins.

[0127] Table 1

[0128]

[0129] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the following claims indicating the true scope and spirit of the disclosure.

Claims

1. A method for purifying a dulaglutide composition, the method comprising: loading the dulaglutide composition together with the buffer B solution onto a hydrophobic interaction chromatography (HIC) column online in a downstream direction, wherein the dulaglutide from the dulaglutide composition binds to the HIC column; Wash the HIC column with a buffer mixture of buffer A solution and buffer B solution; and Dulaglutide was eluted from the HIC column using a linear gradient of percentages of buffer A and buffer B solutions, wherein the linear gradient was followed by an isocratic hold based on UV absorbance readings from the HIC column eluate, indicating elution of protein-containing material from the HIC column, and the isocratic hold was maintained until front cleavage was performed, and then the buffer B solution was reduced to 0% to allow dulaglutide to elute from the HIC column.

2. The method according to claim 1, wherein the dulaglutide composition and the buffer B solution are loaded at a volume ratio of 62.7% to 72.5% of the buffer B solution.

3. The method according to claim 1 or 2, wherein the dulaglutide composition is loaded online with a target loading range of 10 g to 20 g dulaglutide per liter of HIC resin.

4. The method according to any one of claims 1 to 3, wherein the dulaglutide composition is loaded at a linear velocity ranging from 80 cm / hr to 160 cm / hr.

5. The method according to any one of claims 1 to 4, wherein the buffer A solution comprises 20 mM Tris and has a pH range of 7.7-8.

3.

6. The method according to any one of claims 1 to 5, wherein the buffer B solution comprises 20 mM Tris, 400 mM sodium sulfate, and has a pH range of 7.7-8.

3.

7. The method according to any one of claims 1 to 6, wherein the wash buffer mixture comprises 15%-25% of buffer A solution and 75%-85% of buffer B solution.

8. The method according to any one of claims 1 to 7, wherein the wash buffer mixture comprises 20% buffer A and 80% buffer B solution.

9. The method according to any one of claims 1 to 8, wherein the wash buffer mixture comprises 20 mM Tris, 320 mM sodium sulfate solution, and has a pH range of 7.7-8.

3.

10. The method according to any one of claims 1 to 9, wherein washing is performed at a linear velocity of the buffer mixture in the range of 80 cm / hour to 160 cm / hour.

11. The method according to any one of claims 1 to 10, wherein the HIC column is washed with 2.7 column volumes (CV) to 3.3 CV of the buffer mixture.

12. The method according to any one of claims 1 to 11, wherein the HIC column is washed with 3.0 CV of the buffer mixture.

13. The method according to any one of claims 1 to 12, wherein the linear gradient starts at a point in the range of 67.5% to 72.5% of buffer B solution and ends at a point in the range of 5% to 50% of buffer B solution.

14. The method according to any one of claims 1 to 13, wherein dulaglutide is eluted using a linear gradient of 10 column volumes (CV) to 12 CV of buffer A and buffer B.

15. The method according to any one of claims 1 to 14, wherein dulaglutide is eluted with 11.5 CV of buffer A and B solution using a linear gradient.

16. The method according to any one of claims 1 to 15, wherein dulaglutide is eluted with a linear gradient at a linear velocity of 80 cm / hr to 160 cm / hr.

17. The method of any one of claims 1 to 16, wherein initiation of the isocratic hold is based on UV absorbance at A280 nm and a UV signal in the range of 0.1 AU / cm-0.5 AU / cm.

18. The method of any one of claims 1-17, wherein initiation of an isocratic hold is based on UV absorbance at A280 nm and a UV signal at about 0.25 AU / cm.

19. The method according to any one of claims 1 to 17, wherein the isocratic hold is maintained with 1.25 to 1.75 CV of buffer B solution.

20. The method of any one of claims 1-19, wherein the isocratic hold is maintained with about 1.5 CV of buffer B solution.

21. The method of claim 20, wherein the front side cleavage is performed with 0% buffer B solution.

22. The method according to any one of claims 1 to 21, wherein when the front cleavage is performed, collection of the eluate comprising dulaglutide begins.

23. The method of claim 22, wherein the collecting is continued until the UV absorbance reading of the eluate at A280 nm is in the range of 0.125 AU / cm-0.25 AU.

24. The method of claim 23, wherein the acquisition is continued until a UV signal of approximately 0.25 AU / cm is reached.

25. The method of any one of claims 1-24, wherein the dulaglutide composition comprises a plurality of compounds derived from a mammalian fermentation process.

26. The method according to any one of claims 1 to 25, wherein the dulaglutide composition is a downstream product of an anion exchange chromatography column.

27. The method of any one of claims 1-26, wherein the plurality of compounds comprises a host cell protein.

28. The method of any one of claims 1 to 27, wherein the HIC column comprises a highly cross-linked agarose matrix with phenyl ligands.

29. The method of any one of claims 1 to 28, wherein the HIC column has a diameter in the range of 50 cm to 200 cm.

30. The method of any one of claims 1 to 29, wherein the HIC column has a packed bed height in the range of 15 cm to 30 cm.

31. The method of any one of claims 1 to 30, wherein the HIC column comprises one or more upstream in-line filters.

32. The method of any one of claims 1-31, wherein the HIC column comprises two 0.2 micron filters upstream of the HIC column.

33. The method of any one of claims 1 to 32, further comprising equilibrating the HIC column prior to loading using a 2 CV linear gradient of 32.5% buffer A and 67.5% buffer B in a downstream direction.

34. The method of any one of claims 1-33, wherein eluting further comprises initiating a mainstream product collection step by performing a front-side cut and terminating the mainstream product collection step by performing a back-side cut at a UV absorbance of 0.125 AU / cm or greater at A280 nm.

35. The method according to any one of claims 1 to 34, further comprising one or more of the following steps: regenerating the HIC column, disinfecting the HIC column, and storing the HIC column.

36. The method of claim 35, wherein regenerating the HIC column comprises washing the HIC column with 3.0 CV of 0.01 N NaOH in an upflow direction.

37. The method of claim 35 or 36, wherein regeneration of the HIC column occurs after each elution step.

38. The method of claim 35, wherein sanitizing the HIC column comprises washing the HIC column with 2.0 CV of 1 N NaOH in an upflow direction, wherein the target contact static hold time is no less than 30 minutes.

39. The method of claim 35, wherein storing the HIC column comprises washing the HIC column in an upstream direction using at least 2 CV of 0.01 N NaOH storage buffer solution when the HIC column is maintained for greater than 24 hours prior to loading the dulaglutide composition.

40. A method for purifying a dulaglutide composition, the method comprising: loading a dulaglutide composition and a buffer B solution onto a hydrophobic interaction chromatography (HIC) column online in a downstream direction at a volume ratio ranging from 62.7% to 72.5% and a linear velocity ranging from 80 cm / hr to 160 cm / hr, wherein dulaglutide from the dulaglutide composition binds to the HIC column, and the composition comprises a plurality of compounds derived from a mammalian cell fermentation process; washing the HIC column with a buffer mixture comprising 15% to 25% of a buffer A solution and 75% to 85% of a buffer B solution in a downstream direction at a linear velocity range of 80 cm / hour to 160 cm / hour for a period of 2.7 column volumes (CV) to 3.3 CV; and Dulaglutide was eluted from the HIC column using a linear gradient comprising 67.5% of buffer B solution to 10% of buffer B solution for a period of 10 CV to 12 CV, wherein the linear gradient was followed by an isocratic hold based on UV absorbance readings of the eluate at A 280 nm in the range of 0.1 AU / cm to 0.5 AU / cm, indicating elution of protein-containing material from the HIC column, the isocratic hold using a fixed % of buffer B solution was maintained for 1.25 to 1.75 CV, a front-side cut was performed using 0% buffer B solution, the eluate containing dulaglutide was collected until the UV absorbance reading at A 280 nm was in the range of 0.125 AU / cm to 0.25 AU, and a back-side cut was performed when the UV absorbance reading of the eluate at A 280 nm was 0.125 AU / cm to 0.25 AU.

41. The method of claim 40, wherein the HIC column comprises a highly cross-linked agarose matrix with phenyl ligands.

42. The method of claim 40 or 41, wherein the HIC column has a diameter in the range of 50 cm to 200 cm.

43. The method of any one of claims 40 to 42, wherein the HIC column has a packed bed height in the range of 15 cm to 30 cm.

44. The method of any one of claims 40-43, wherein the HIC column comprises one or more upstream in-line filters.

45. The method of any one of claims 40-44, wherein the HIC column comprises two 0.2 micron filters upstream of the HIC column.

46. ​​The method of any one of claims 40-45, further comprising in-line blending the composition with a buffer B solution via a loading and buffer supply pump prior to loading.

47. The method of any one of claims 40-46, wherein the plurality of compounds in the composition comprises host cell proteins.

48. The method of any one of claims 40-47, wherein the plurality of compounds in the composition comprises a reversed host cell protein.

49. The method of claim 48, wherein the purification process reduces the concentration of host cell protein in the composition from the range of 30-70 ppm to about 10 ppm.

50. The method according to any one of claims 40 to 49, wherein the concentration of dulaglutide present in the mainstream product after elution from the HIC column is in the range of 2.15 g / L to 6.51 g / L.

51. The method of claim 50, wherein the concentration of host cell protein in the mainstream product after elution from the HIC column is in the range of 2 ppm to 11 ppm.

52. Dulaglutide prepared by the method according to any one of the preceding claims.

53. A composition comprising dulaglutide prepared by the method according to any one of the preceding claims.

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