Virus filtration of therapeutic antibodies through reconstitution of desirable elution sub-pools

Abstract

Methods for fractionating mAbs into sub-pools, comprising steps for introducing an equilibration buffer in a column, loading a mAb solution at desirable buffer conditions into a chromatography column; optionally washing the chromatography column; introducing an elution buffer into the chromatography column; starting a step pH gradient elution at 10 percent buffer B and increasing to 40 percent buffer B or starting a step pH gradient elution at 40 percent buffer B and decreasing to 10 percent buffer B, wherein buffer B operates in a neutral or basic pH range; collecting a plurality of sub¬ pools of mAb proteoforms at a given step pH value, wherein each sub-pool is enriched with different proteoforms of mAb; optionally characterizing the plurality of sub-pools; optionally reconstituting the plurality of sub-pools into a single pool comprising two or more proteoforms; and optionally filtering the single pool are disclosed.

Description

Attorney Docket No.: P25-033-SEC-W001VIRUS FILTRATION OF THERAPEUTIC ANTIBODIES THROUGH RECONSTITUTION OF DESIRABLE ELUTION SUB-POOLSRelated Applications

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 755,049, filed February 6, 2025, the entire content of which is incorporated herein by reference.Field

[0002] Methods and systems for the processing of biological fluids are disclosed. More particularly, embodiments described herein relate to systems and processes for methods for improving filtration of biological fluids via reconstitution of elution sub-pools using chromatography media.BACKGROUNDRelated Art

[0003] Processes for growing, formulating cell cultures, and harvesting live cellular derived products, such as monoclonal antibodies, cells, viruses, and other biological products, are difficult to develop. Many critical attributes and properties, which impact cell growth and productivity, must be monitored and controlled. These upstream processes generate contaminants, including biological contaminants, such as viruses and bacteria, which must be purified by downstream processes. Purification processes include clarification filters, concentration and diafiltration using tangential flow filtration, chromatography purification by using affinity chromatography and ion exchange chromatography and / or the like. In some processes, ultracentrifugation and gradient ultracentrifugation are used instead of chromatography or in addition to chromatography. Furthermore, the filters used are typically single-use and become fouled, necessitating redundant filtration or purification steps. Accordingly, these filtration and purification operations are time-intensive and expensive.

[0004] Physicochemical properties play a significant role in the manufacturability of drug candidates. Properties such as solubility, permeability, and chemical stability can greatly impact the formulation and process development of drugs. Properties such as solubility, permeability, and chemical stability can significantly influence formulation and processAttorney Docket No.: P25-033-SEC-W001development. While poor solubility can pose challenges in formulating an effective dosage form, instability under certain conditions may necessitate special purification and storage requirements. A thorough evaluation of mAb properties enables valuable insights into the mAb's compatibility with manufacturing processes and formulation approaches.

[0005] Several researchers have published methods for charge variant fractionation of monoclonal antibodies (mAbs), especially using analytical high-performance liquid chromatography (HPLC) columns for qualitative assessment of product quality. Some methods have used analytical HPLC columns, such as weak cation exchange or a size exclusion column (SEC) demonstrating correlations between acidic, neutral, and basic charge variants and aggregate percentage on an SEC column. These generally show aggregates that are enriched in the basic species. Other studies have demonstrated semi-preparative scale separation of charge variants using a cuboid packed bed device. Such approaches are limited to qualitative assessment of product variants after semi-preparative fractionation. Some other studies have separated a well-behaved monoclonal antibody into proteofo rm -enriched sub-pools at large scale. This body of work consists of highly hydrophobic, difficult-to-filter molecules, wherein the fractionated mAb isoforms are reconstituted based on the stability and filterability of each fractionated sub-pool with the goal of obtaining a more filterable pool or batch than the bulk unfractionated feed mAb. Another method uses cation exchange media in flowthrough mode to enrich post-translational modifications. This approach consists of a single eluate pool having a different profile from the feed pool. For example, two buffers may be used in such an elution. A first buffer is a low conductivity / ionic strength salt, e.g., 20 millimolar salt solution, a second buffer is a high conductivity, e.g., 1.0 Molar, salt solution. During processing, the pH may be increased by 0.1 pH units over several minutes, wherein the binding of mAbs occurs indiscriminately and different proteoforms are enriched within a single eluate sub-pool.

[0006] Novel paradigms in mAb developability use bioanalytical characterization to address manufacturability questions such as aggregation and stability characteristics of the biologic especially in the context of virus filtration and prefiltration by design. Methods and cell culturing apparatus forAttorney Docket No.: P25-033-SEC-W001producing antibodies, therapeutic proteins, and other biological products that balance processing expense, quality and concentration while providing increased product recovery represent an advance in the art. The inventors have surprisingly discovered innovative methods and apparatus, employing mixed mode resins in bind and elute mode to fractionate multiple mAb proteoforms, demonstrating significant processability of various sub-pools on the virus filtration step(s), without the significant fouling of filters, representing inventive advances in the art.SUMMARY

[0007] Methods according to some embodiments of the disclosure comprise methods forfractionating mAbs into sub-pools, comprising steps for introducing an equilibration buffer to a column followed by a mAb solution; loading the mAb solution at a desired buffer condition into a chromatography column; optionally washing the chromatography column; loading an elution buffer into the chromatography column; starting a step pH gradient elution at 10 percent buffer B and increasing to 40 percent buffer B or starting a step pH gradient elution at 40 percent buffer B and decreasing to 10 percent buffer B, wherein buffer B operates in an acidic or basic range; collecting a plurality of sub-pools containing mAb proteoforms at a given step pH value, wherein each sub-pool is enriched with different proteoforms of mAb; optionally characterizing the plurality of sub-pools; optionally reconstituting the plurality of sub-pools into a single pool comprising two or more proteoforms; and optionally filtering the single pool are disclosed.

[0008] Preparative scale mAb fractionation to enrich undesirable high mannose glycoforms and other aggregation-prone proteoforms into sub-pools before reconstituting more desirable post-elution sub-pools into a less fouling batch for VPro virus filters; substantially as shown in and / or described in connection with at least one of the figures, as set forth more completely in the claims, are disclosed. In some embodiments, systems described herein comprise methods and systems for chromatographically fractionating a bulk mAb pool into isoform and glycoform enriched sub-pools for enriched microvariant manufacturability assessment analysis, allowing the acquisition of multiple manufacturability datasets that correspond to the relative amounts of different proteoforms or glycoforms in each sub-pool. This sub-pool specific information can help predictAttorney Docket No.: P25-033-SEC-W001problematic isoforms and glycoforms to selectively capture these species. These methods permit the consolidation of enriched isoform sub-pools for bioanalytical characterization and manufacturability assessment. Recognizing the stability and aggregation susceptibility of each sub-pool permits the early identification of unmanufacturable sub-pools, allowing the provision of feedback to upstream and drug development processing. Bioanalytical assays of interest at this stage of sub-pool developability further include diffusion interaction parameter (kD), relative hydrophobicity, oligomericity using size exclusion chromatography, glycan profile and charge variant or imaged capillary isoelectric point profile. In some embodiments, the chromatography column used during processing of the mAbs comprise cationic ligands, i.e., carboxylic acid ligands in conjunction with hydrophobic ligands. In some embodiments, multiple proteoforms are separated into discrete sub-pools of proteoforms. In some embodiments, a specific proteoform-enriched pool is filtered. In some embodiments, a plurality of proteoforms is separated from a batch and reconstituted before filtration, wherein the highly aggregating proteforms are removed before filtration. In this context, highly aggregating mAbs are considered to possess ko values of -8 mL per gram or less (for example -9 or -20 mL per gram), while low-aggregating mAbs are considered to possess ko values exceeding -3 mL per gram (for example -1 or+1 mL per gram). In some embodiments, the first proteoform-enriched sub-pool eluted and the last proteoforms-enriched sub-pool eluted are removed and the remaining subpools are subjected to filtration, e.g., virus filtration. In some embodiments, the most hydrophobic proteoform(s) is / are removed via pH step gradient elution before filtration, e.g., virus filtration. In some embodiments, between one and five; or one and ten; or between one and fifteen sub-pools of proteoforms are separated during elution. In some embodiments, between two and 15 chromatographic peaks are created, representing, respectively, two to fifteen proteoform-enriched fractions or sub-pools during elution. In some embodiments, various proteins are separated or fractionated by charge variant properties and / or hydrophobic variant properties during elution.Attorney Docket No.: P25-033-SEC-W001BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 depicts a flowchart for a first method for fractionating mAb and filtering using virus filters, according to some embodiments of the disclosure;

[0010] FIG. 2 depicts a flowchart for a second method for fractionating mAb and filtering using virus filters, according to some embodiments of the disclosure;

[0011] FIGS. 3A-3E depict various glycovariants, G0F, G1 Fa, G0F-N, G2F, and Mannose 5 in various percentages as collected in a plurality of eluate subpools, when subjected to the conditions and methods of FIG. 1; according to embodiments of the disclosure;

[0012] FIG. 4 depicts the filtration data of 10 g / L, unfractionated mAb 12 in pH 5.5, 100 mM acetate buffer, 10 mS / cm when filtered across a Viresolve Shield prefilter coupled to a Viresolve Pro virus filter; according to embodiments of the disclosure;

[0013] FIG. 5 depicts the volumetric throughput values for each fractionated sub-pool of mAb 12 where higher throughput values are obtained in comparison with the feed mAb fraction; according to embodiments of the disclosure;

[0014] FIG. 6 depicts the chromatographic fractionation of mAb 12 using cation exchange chromatography resin, Eshmuno® CPX, used to fractionate isoformenriched sub-pools of mAb 12 from step 104 of the method of FIG. 1 , showing excellent resolution; according to embodiments of the disclosure;

[0015] FIG. 7 depicts the chromatographic fractionation of mAb 12 using mixed mode chromatography resin, Eshmuno® CMX, used to fractionate isoformenriched sub-pools of mAb 12 from step 104 of the method of FIG. 1 , showing excellent resolution; according to embodiments of the disclosure; and

[0016] FIG. 8 depicts the chromatographic fractionation of mAb 12 using Eshmuno® Q anion exchange chromatography resin, used for bind and elute pH gradient fractionation of mAb 12 into sub-pools using a reverse pH gradient from pH 9 to pH 7 of step 206 of the method of FIG. 2; according to embodiments of the disclosure.DETAILED DESCRIPTION OF SOME EMBODIMENTS

[0017] So the manner in which the features disclosed herein can be understood in detail, a more particular description of the embodiments of the disclosure,Attorney Docket No.: P25-033-SEC-W001briefly summarized above, may be had by reference to the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its scope, for the embodiments described and shown may admit to other equally effective embodiments. It is also to be understood that elements and features of one embodiment may be found in other embodiments without further recitation and that identical reference numerals are sometimes used to indicate comparable elements that are common to the figures.

[0018] Purification means to increase the degree of purity of a target molecule, e.g., mAbs, by removing one or more impurities.

[0019] As used herein, the term “proteoform” means a glycoprotein variant that displays microheterogeneities in amino acid sequence modifications or appended glycan type.

[0020] As used herein, the term “proteoform-enriched” means a process pool or fraction with a higher degree of specific proteoforms being enriched and less of certain proteoforms.

[0021] As used herein, the term “fractionated mAb isoform” means a specific protein variant which has been selectively enriched in each process pool or fraction.

[0022] As used herein, the term “sub-pools” means wherein the eluate pool is collected in fractions based on step elution conditions and wherein each eluate fraction is specifically referred to as a sub-pool collected during pH step gradient elution.

[0023] As used herein, the term “eluate pool” means the entirety of the elution fractions and is the sum of all sub-pools.

[0024] As used herein, the term “flowthrough feed pool” means the fraction of the feed material flowing through the chromatography media. In cation exchange bind and elute chromatography, when the buffer pH is less than the isoelectric point of the mAb, the flowthrough fraction comprises impurities while the eluate fraction is the desired product. Conversely, in flowthrough anion exchange polishing chromatography, where the buffer pH is less than the mAb isoelectric point, the flowthrough fraction is the desired product while the eluate fraction comprises impurities.Attorney Docket No.: P25-033-SEC-W001

[0025] As used herein, the term “well-behaved monoclonal antibody” means the diffusion interaction parameter (KD) value to show filterability of a mAb, wherein KD has a positive value or negative value very close to zero (> -3 ml_ / g), the molecule is well-behaved. The Ko-diffusion interaction parameter can be defined as the propensity of self-association of monomeric mAbs or reversible aggregation propensity. A Ko-diffusion interaction parameter value that is highly negative across all sub-pools indicates a high tendency for intermolecular attraction.

[0026] As used herein, the term “aggregation-prone proteoforms” means specific proteoforms of a mAb which have a highly negative KD value (< -8 mL / g) and as a result demonstrate a high self-association and reversible aggregation tendency. It also includes denatured mAb variants which are highly prone to self-association and aggregation.

[0027] As used herein, the term “high mannose glycoform” means a glycoprotein variant which are appended with mannose-5 glycans, mannose 6 glycans or higher mannose glycans such as mannose-8. As used herein, the term “glycovariant” refers to mAb (glycoprotein) variants which possess a different glycosylation pattern such as mannose 5, G0F glycan or any other glycan type. As used herein, the term “glycan” means a polysaccharide that may attach to a protein, for e.g., sometimes at an oxygen or nitrogen atom of a protein. Glycosylation is a process by which a carbohydrate is attached to a target molecule or macromolecule.

[0028] As used herein, the term “post-translational modifications” means enzyme or environmentally mediated processes which occur after protein or mAb expression, typically resulting in a host of micro-heterogeneous product variants specifically known as proteoforms (a subset of isoforms). Some post translational modifications, such as glycosylation, can also result in macroheterogeneities for instance when there is a difference in glycan occupancy region (isoforms) in contrast with glycan type differences that result in glycoform variants.

[0029] As used herein, the term “highly hydrophobic” means a relative value of mAb hydrophobicity when benchmarked against a standard, relatively hydrophobic reference mAb (mAb 05). By benchmarking mAb elution time under similar buffer conditions from an analytical phenyl or butyl resin packedAttorney Docket No.: P25-033-SEC-W001column, a mAb with twice the retention time (minutes) of mAb 05 is referred to as a highly hydrophobic mAb.

[0030] Capillary isoelectric focusing (clEF) is a bioanalytical technique that allows quantitative, experimental determination of the isoelectric focusing point (pl) and charge variants of a protein.

[0031] Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is a bioanalytical technique that allows quantitative, experimental analysis of the molecular weight variants of a protein or protein fragments.

[0032] Size exclusion chromatography (SEC) indicates an analytical chromatography process for molecules to be separated by differences in size.

[0033] The isoelectric point (pl) is the pH condition where a molecule has a net neutral surface charge. It corresponds to the pH of an aqueous solution of biomolecules where the biomolecules on average have no net charge. In other words, the positively charged groups are balanced by the negatively charged groups.

[0034] As used herein, the term “ultracentrifugation” means a very fast centrifugal unit operation used to precipitate large biological molecules from solution or separate them by their different rates of sedimentation. An ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 1 ,000,000 g (approx. 9800 km / s2). There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge. In some cases, an ultracentrifuge processes biofluids at 50,000 to 150,000 revolutions per minute (RPM).

[0035] As used herein, the term “gradient ultracentrifugation” means a widely used method for isolating and purifying biomolecules and cellular structures. This technique takes advantage of the principle that particles denser than the solvent will settle, whereas less dense particles will remain afloat. A high-speed ultracentrifuge accelerates this process, allowing for the separation of biomolecules within a density gradient created by layering liquids of varying densities in a centrifuge tube. Centrifugation can, e.g., be a low-speed centrifugation to remove larger particles, such as cellular debris.

[0036] As used herein, the term “size exclusion column (SEC) demonstrating correlations between acidic, neutral, and basic charge variant-enriched subpools having differing aggregate percentages on an SEC column” means theAttorney Docket No.: P25-033-SEC-W001condition of a mAb feed having been fractionated into eluate sub-pools using a desirable chromatography resin packed column, wherein the eluate sub-pools show differences in the percentage of acidic charge variants, main charge variants and neutral charge variants that usually directly correlates with the percentage of higher molecular weight species (aggregates) in each sub-pool. Aggregates are typically depleted in the acidic charge variant enriched subpools whereas aggregates tend to be enriched in the sub-pools with a high degree of basic charge variants.

[0037] The term "chromatography" refers to any kind of technique which separates an analyte of interest, a target molecule, from other molecules present in a sample. Usually, the target molecule, e.g., mAb, is separated from other molecules because of differences in rates at which the individual molecules of the mixture bind to and / or migrate through a chromatography matrix under the influence of a mobile phase.

[0038] The term "matrix" or "chromatography matrix" are used interchangeably herein and refers to a solid phase through which the sample migrates during a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material.

[0039] A “ligand” is a functional group that is part of the chromatography matrix, typically attached to the base material of the matrix, and that determines the binding properties and interaction properties of the matrix.

[0040] Matrices of the disclosure comprise at least cation exchange groups and hydrophobic interaction groups. It may be a mixed mode cation exchange chromatography matrix. The cation exchange groups may, for example, be strong cation exchange groups, such as sulfonic acid groups. They may also be weak cation exchange groups, such as carboxymethyl or carboxylic acids. Examples of hydrophobic interaction groups are hydrophobic groups, e.g., phenyl, butyl, propyl, hexyl. The matrix may comprise two or more different cation exchange groups, e.g., weak and strong cation exchange groups. Matrices may also comprise two or more different hydrophobic interaction groups. Any of the matrices may additionally comprise other types of ligands. The groups may be part of the base material or, alternatively, may be part of a ligand. One ligand may comprise one or several different cation exchange and / or hydrophobic groups.Attorney Docket No.: P25-033-SEC-W001

[0041] The term "impurity" or “contaminant” as used herein, refers to any foreign or objectionable molecules or species, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, viruses, bacteria, endotoxins, lipids, impurities of synthetic origin like detergents, partial and / or empty adeno-associated viruses (AAVs) as well as one or more additives which may be present in a sample containing the mAb particles to be purified and thus to be separated from one or more of the impurities.

[0042] As used herein, and unless stated otherwise, the term “sample” may comprise any biological fluid composition or mixture. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues, and organs. The sample may also include diluents, buffers, detergents, and contaminating species, adjuvants, salts, excipients, debris and the like that are found mixed with the target molecule. The sample may be "partially purified" (i.e., having been subjected to one or more purification steps, e.g., filtration steps) or may be obtained directly from a host cell producing the target molecules, e.g., the sample may comprise harvested cell culture fluid.

[0043] A "buffer" is a solution that resists changes in pH by the action of its acidbase conjugate components. For example, a weak acid and its conjugate base, or a weak base and its conjugate acid. One such buffer is acetic acid and sodium acetate, which is effective in the acidic pH range. A “Tris” buffer, e.g., Tris(hydroxymethyl)ami nomethane, is capable of maintaining a stable pH level typically within the range of 7-9, making it suitable for various biological processes like protein purification, cell culture, and nucleic acid manipulation during the production of biopharmaceuticals.

[0044] When “loading” a chromatography column in bind and elute mode, the sample or composition comprising the target molecule and one or more impurities is loaded onto a chromatography column. In preparative chromatography, the sample is preferably loaded directly without the addition of a loading buffer. If a loading buffer is used, the buffer has a composition, a conductivity and / or pH such that the target molecule is bound to the stationary phase while ideally all the impurities like the host cell proteins or molecules are not bound and flow through the column. Typically, the loading buffer, if used, has the same or similar composition as the equilibration buffer used to prepare the column for loading.Attorney Docket No.: P25-033-SEC-W001

[0045] The composition of the sample loaded on the chromatography column is called “feed.” The feed may comprise the sample and the loading buffer but typically comprises the sample. A fluid entering a chromatography column is called an “eluent,” while the fluid exiting the chromatography column, i.e., undergoes “elution,” is called an “eluate.”

[0046] To "elute" a molecule (e.g., the target molecule) from a matrix means that the molecule is removed therefrom. Elution may take place by altering the solution conditions such that a buffer different from the loading and / or washing buffer competes with the molecule of interest for the ligand sites on the matrix or alters the equilibrium of the target molecule between stationary and mobile phase such that the target molecule is favored and is preferentially present in elution buffer.

[0047] The terms “flow-through process,” “flow-through mode,” and “flow-through operation,” as used interchangeably herein, refer to a chromatographic process in which at least one target molecule (e.g., a protein) contained in a sample along with one or more impurities is intended to flow through a chromatography matrix, which usually binds one or more impurities, where the target molecule usually does not bind (i.e., flows through) and is eluted from the chromatograph matrix with the loading buffer.

[0048] The terms "bind and elute mode" and "bind and elute process," as used herein, refer to a separation technique in which at least one target molecule contained in a sample (e.g., a protein) binds to a suitable chromatography matrix and is subsequently eluted with a buffer different from the loading buffer.

[0049] The embodiments described herein are further illustrated by the following figures and examples which are illustrative, however, without being restricted thereto.

[0050] Some embodiments of the disclosure comprise a method for enhanced prefiltration and virus filtration of mAb glycoforms or isoforms demonstrating minimal fouling with targeted prefiltration of any difficult-to-filter sub-pools. Some embodiments of the disclosure comprise converting a difficult-to-filter biological fluid comprising mAbs into a more filterable biological fluid that can be filtered using standard filtering techniques. Novel methods of some embodiments comprise the use of a chromatography resin packed in a Vantage column for large scale glycovariant and isoform fractionation into sub-pools withAttorney Docket No.: P25-033-SEC-W001varying hydrophobicity, reversible aggregation propensity (ko value), and higher molecular weight ratios. Such a resin comprises weak cation exchange properties and hydrophobic interaction, providing high selectivity for monoclonal antibody (mAb), fusion protein and Antibody Drug Conjugate (ADC) purification as well as separation of low molecular weight impurities and Host Cell Proteins (HCPs). One such resin is Eshmuno® CMX, manufactured and marketed by EMD Millipore Corporation, Burlington, MA, USA. Eshmuno® CMX is a mixed-mode resin, comprising cationic exchange (CEX) ligands and Hydrophobic-Interaction Chromatography (HIC) ligands, featuring weak cation exchange properties with hydrophobic interaction, providing high selectivity for Monoclonal Antibody (mAb), fusion protein and Antibody Drug Conjugate (ADCs) purification as well as separation of low molecular weight impurities, Host Cell Proteins (HCPs) and glycovariants. Eshmuno® CMX is a chromatography resin having ligands comprising COO' and alkyl groups. Critical quality attributes of each sub-pool are used to reconstitute desirable sub-pools into feed mAb for the Viresolve ® Pro virus filter, manufactured and marketed by EMD Millipore Corporation, Burlington, MA, USA, for reduced fouling and better volumetric throughput.

[0051] In a first method, Eshmuno® CMX mixed mode resin (cation exchange plus hydrophobic interaction chromatography) was packed into a, for example, Vantage column to a bed height of 18.5 cm (Vantage® L Laboratory Column, 1.6 cm column ID x 25 cm column height). Vantage L columns are manufactured and marketed by EMD Millipore Corporation, Burlington, MA, USA. Other columns are acceptable for methods described herein. When packed with Eshmuno CMX resin, these columns can be used to fractionate monoclonal antibody (mAb) glycoforms and isoforms, in bind and elute mode, into sub-pools enriched with different variants for assessment of filterability of each sub-pool via one or more virus filtration operations. The sub-pools can be reconstituted into a bulk pool, wherein the desirable sub-pools for improved virus filtration are filtered. The test mAb having an Isoelectric point (pl) value of 8.2 binds to the column at a pH of 7.0 and a step gradient is run from pH 7.0 to a pH of 9.0 (at 8 mL / min corresponding to a residence time of four minutes) and conductivity from 1.4 mS / cm to 4.2 mS / cm.Attorney Docket No.: P25-033-SEC-W001

[0052] All experiments were conducted at or near room temperature. A polished mAb feed was prepared in a low conductivity buffer (25 millimolar (mM) sodium acetate plus acetic acid, pH 5.6, conductivity 2 mS / cm). The chromatography column parameters are as follows: 37 mL Vantage L column (1.6 cm inner diameter and 18.5 cm packed bed height). Elution step lengths were equal to 2 column volumes (CVs). A linear velocity across column was 240 centimeters per hour (cm / hr), a flowrate was 8 milliliters per minute (mL / min) and a mAb concentration was 2.5 grams per liter (g / L). An Equilibration buffer solution A, 25 mM Tris base titrated with glacial acetic acid (pH 7, conductivity 1.4 (milliSiemens per centimeter (mS / cm) was used. An elution buffer B was 35 mM sodium chloride, and a 50 mM tris base titrated with glacial acetic acid (pH 9, conductivity 4.2 mS / cm) were used.

[0053] Eluate sub-pools were independently assessed using virus filters, e.g., Viresolve® Pro Micro 40 virus filters (3.4 cm2filtration area) and the worst performing sub-pools were eliminated from the reconstituted sub-pools to yield better filtration outcomes. In this context, “better” filtration outcomes indicates that there was greater than a 2X increase in final filtered volume before complete fouling of the virus filter. The worst performing sub-pools may be held in a holding tank for more rigorous process development approaches.

[0054] A step pH gradient elution produced optimal separation and well-resolved peaks for acidic, main, and basic charge variants of mAb. In this context, “acidic” indicates a charge variant having a lower isoelectric point than the main variant, wherein “main” indicates charge variants with an average nominal isoelectric point across all charge variants and “basic” means charge variants with an isoelectric point above the isoelectric point of main charge variants. Glycovariants were also enriched in different sub-pools in order of relative isoelectric point of glycoforms. At least one advantage of a multimodal cation exchange resin is the ability to leverage both cationic and hydrophobic chemistry for improved resolution of the mAb isoforms. This method consolidates enriched isoform sub-pools for bioanalytical characterization and manufacturability assessment.

[0055] Without intending to be limited by theory, it is believed that the fractionation of biological fluids into eluate sub-pools enables the enrichment of more filterable variants in some sub-pools and depletion of more aggregation-Attorney Docket No.: P25-033-SEC-W001prone variants in specific sub-pools. The aggregation-susceptibility of different mAb variants (isoforms) is the basis for determining which sub-pools are ideal for reconstitution, i.e., separate sub-pools having less aggregation. The filtration behavior of different sub-pools on the virus filter also validates this theory and is a second basis for determining sub-pools to reconstitute.

[0056] For example, at least one embodiment of the disclosure describes a first method for optimizing a bind and elute step, buffer conditions, and elution step lengths for bulk mAb fractionation. Step gradients are designed to result in pH increments of 0.1 pH unit per step and operate at this condition until two column volumes of elution buffer are pumped across the column, for example, cation exchange or mixed mode chromatography fractionation of mAb 12 and reconstitution of eluate sub-pools for virus filtration. FIG. 1 depicts a flowchart for a first method 100 for fractionating mAbs using cation exchange or mixed mode resins including Eshmuno® CPX and Eshmuno® CMX. The method 100 starts and at step 102, a mAb solution in the desired buffer condition is provided. At step 104, an equilibration buffer is introduced to the column before mAb solution is loaded into the chromatography column. An optional wash step may be employed at this time. At step 106, an elution buffer is introduced into the chromatography column. At step 108, a step pH gradient starts at 10 percent buffer B and increases to 40 percent buffer B. During step 110, during elution at a given step condition (given pH value), a sub-pool(s) of mAbs, e.g., mAb 12, is / are collected such that as many sub-pools as there are different elution steps are collected, e.g., 13% buffer B, 16% buffer B, 19% buffer B, etc., to 40% buffer, for collecting one sub-pool at each elution step. Each sub-pool is enriched with different proteoforms of mAb 12. The buffer exchange step is used to exchange the buffer condition of each sub-pool into a single buffer condition for all sub-pools to maintain uniformity in concentration, pH, and conductivity during bioanalytical characterization. At step 112 optionally, bioanalytical characterization (s) is / are used to obtain relative values for hydrophobicity, diffusion interaction parameter (ko), capillary isoelectric focusing (clEF), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC) and glycan profile for the sub-pool(s).

[0057] Following characterization, sub-pools that are susceptible to aggregation, which causes fouling of filters, may be removed, and set aside forAttorney Docket No.: P25-033-SEC-WG01enhanced filtration strategies. The sub-pools comprising proteoforms that are least susceptible to aggregation can then be optionally re-constituted at step 114 into a single pool or multiple sub-pools and optionally filtered at step 116, for example, filtered to remove viruses. Because the highly aggregating proteoforms were removed, the virus filters used can be employed for much longer durations and / or volumes. The graph below shows the glycan profiles of different sub-pools from Eshmuno® CMX fractionation of mAb 12. Stability and aggregation susceptibility of each sub-pool may offer a fast-track technique for early identification of unmanufacturable sub-pools for feedback to upstream and drug development teams. As shown, various glycans, GOF, G1Fa, GOF-N, G2F, and Mannose 5 are shown in various percentages as collected in a plurality of eluate sub-pools, when subjected to the conditions and methods of the method 100.

[0058] Rapid fouling during virus filtration of mAb 12; virus filtration (VF) performance is a metric for manufacturability of mAbs, i.e., aggregation sensitivity. The buffer exchanged sub-pools with different bioanalytical attributes are loaded on the virus filters, e.g., Viresolve® Pro virus filter, to determine filterability of each proteoform-enriched sub-pool. The figure below shows the rapid fouling of Viresolve® Pro virus filter during the filtration of the bulk mAb 12 feed fraction, prior to chromatographic fractionation, where the final filtrate throughput is 3.5 L / m2, which falls far short of the target throughput.

[0059] The subsequent graph below shows the volumetric throughput values for each fractionated sub-pool of mAb 12 where higher throughput values are obtained in comparison with the feed mAb fraction above. Sub-pool A is the first eluting sub-pool and sub-pool I is the last eluting sub-pool. Eluate sub-pools have different glycoform and isoform profiles and filter differently on a virus filter.

[0060] At least one other embodiment of a method according to the disclosure recites mAb fractionation using ESHMUNO® ion exchange (IEX) resins, manufactured by the EMD Millipore Corporation, Burlington, MA, USA. One such resin is ESHMUNO® CMX, which is a mixed mode chromatography resin combining weak cation exchange properties with hydrophobic interactions.

[0061] In the first and second methods, 100 and 200, according to some embodiments of the disclosure described herein, a mAb 12, nominal pl ~ 8.2,Attorney Docket No.: P25-033-SEC-W001is subjected to either Eshmuno® CPX cation exchange (CEX) resin, Ligand: SO3 ' Eshmuno® CMX mixed-mode (CEX + HIC) resin, Ligand: COO-+ alkyl groups, Eshmuno® Q anion exchange (AEX) resin, Ligand: T rimethylammoniumethyl (TMAE). As shown in FIG. 2, a method 200 starts and at step 202, a mAb solution in a desirable buffer condition is provided. In some embodiments, the second method comprises anion exchange chromatographic fractionation of mAb 12 and reconstitution of eluate sub-pools for virus filtration. At step 204, an equilibration buffer is introduced to the column followed by the mAb solution which is loaded into the chromatography column. An optional wash step may be employed at this time. At step 206, an elution buffer is introduced into the chromatography column and pH is gradually decreased according to the protocol. At step 206, a step pH gradient starts at 40 percent buffer B and decreases to 10 percent buffer B. During step 208, during elution at a given step condition (given pH value), a sub-pool(s) of mAbs, e.g., mAb 12, is / are collected such that as many sub-pools as there are different elution steps are collected, e.g., 40% buffer B, 37% buffer B, 34% buffer B, etc., to 10% buffer B, one sub-pool is collected at each elution step. Each sub-pool is enriched with different proteoforms of mAb 12. The buffer exchange step is used to exchange the buffer condition of each sub-pool into a single buffer condition for all subpools to maintain uniformity in concentration, pH, and conductivity for bioanalytical characterization. At step 210, optionally, bioanalytical characterization(s) is / are used to obtain relative values for hydrophobicity, diffusion interaction parameter (ko), capillary isoelectric focusing (clEF), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC) and glycan profile for the sub-pool(s). Following characterization, sub-pools that produce more aggregates, which cause fouling of filters, may be removed, and set aside. The sub-pools comprising proteoforms that are less susceptible to aggregation can then be optionally reconstituted at step 212 into a single pool or multiple sub-pools and optionally filtered at step 214, for example, filtered to remove viruses. Because the highly-aggregating proteoforms were removed, the virus filters used can be employed for much longer durations and / or volumes. In the first step 202, mAbs, such as mAb 12, is loaded on a Vantage column packed with Eshmuno® CPX and eluted from pH 7 to pH 9 to obtain proteoform-enriched sub-pools as describedAttorney Docket No.: P25-033-SEC-W001for Eshmuno® CMX under identical buffer and formulation conditions. Eshmuno® CPX is a single mechanism cation exchange resin.

[0062] In the step 204, Eshmuno® CMX is used to fractionate isoform-enriched sub-pools of mAb 12 which shows excellent resolution and is already described in detail above.

[0063] In the step 206, Eshmuno® Q anion exchange resin is used for bind and elute pH gradient fractionation of mAb 12 into sub-pools using a reverse pH gradient from pH 9 to pH 7, unlike the cation exchange resins which use a forward pH gradient.

[0064] FIG. 3 shows a distribution of major glycovariants of mAb 12 across the eluate sub-pools of Eshmuno CMX mixed mode chromatography resin. Specifically, FIGS. 3A-3E depict various glycovariants, G0F, G1Fa, G0F-N, G2F, and Mannose 5 in various percentages as collected in a plurality of eluate sub-pools, when subjected to the conditions and methods of FIG. 1 ; according to embodiments of the disclosure.

[0065] FIG. 4 shows filtration data of 10 g / L, unfractionated mAb 12 in pH 5.5, 100 mM acetate buffer, 10 mS / cm when filtered across a Viresolve Shield prefilter coupled to a Viresolve Pro virus filter.

[0066] FIG. 5 depicts final volumetric throughput of mAb 12 sub-pools filtered through the Viresolve Pro virus filter without prefiltration under uniform formulation conditions (5 g / L mAb 12, sub-pools A-l in 20 mM tris acetate, 5 mM NaCI; pH 8, 1.2 mS / cm).

[0067] FIG. 6 depicts a chromatogram for pH gradient fractionation of mAb 12 using Eshmuno® CPX cation exchange resin.

[0068] FIG. 7 depicts a chromatogram for pH gradient fractionation of mAb 12 using Eshmuno® CMX mixed-mode resin.

[0069] FIG. 8 depicts a chromatogram for pH gradient fractionation of mAb 12 using Eshmuno® Q anion exchange resin.

[0070] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in oneAttorney Docket No.: P25-033-SEC-W001embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment.

[0071] Although some embodiments have been discussed above, other implementations and applications are also within the scope of the following claims. Although the specification describes, with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be further understood that numerous modifications may be made to the illustrative embodiments and that other arrangements and patterns may be devised without departing from the spirit and scope of the embodiments according to the disclosure. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more of the embodiments.

[0072] Publications of patent applications and patents and other non-patent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

Attorney Docket No.: P25-033-SEC-W001CLAIMSWhat is claimed is:

1. A method for fractionating mAbs into sub-pools, comprising:introducing an equilibration buffer to a column followed by a mAb solution in the desired buffer condition;loading the mAb into a chromatography column;optionally washing the chromatography column;introducing an elution buffer into the chromatography column; starting a step pH gradient elution at 10 percent buffer B and increasing to 40 percent buffer B, wherein buffer B operates in a basic pH range;collecting a plurality of sub-pools of mAb proteoforms at a given step pH value, wherein each sub-pool is enriched with different proteoforms of mAb;optionally characterizing the plurality of sub-pools;optionally reconstituting the plurality of sub-pools into a single pool comprising two or more proteoforms; andoptionally filtering the single pool.

2. The method of claim 1 , wherein each sub-pool(s) of mAbs is collected such that as many sub-pools as there are different elution steps are collected at 13% buffer B, 16% buffer B, 19% buffer B, etc., to 40% buffer, for collecting a plurality of sub-pools, wherein each sub-pool is enriched with different proteoforms of mAbs.

3. The method of claim 1 wherein the characterization step evaluates sub-pools for uniformity in concentration, pH, and conductivity.

4. The method of claim 1 , where the bioanalytical characterization(s) characterizes at least one of relative values for hydrophobicity, diffusion interaction parameter (kD), capillary isoelectric focusing (clEF), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC) and glycan profiles.

5. The method of claim 1 , wherein the buffer B comprises tris(hydroxymethyl) aminomethane.

6. The method of claim 1 , wherein the buffer B comprises acetic acid and sodium acetate.

7. The method of claim 1 , wherein a resin within the chromatography column is a mixed-mode resin.Attorney Docket No.: P25-033-SEC-W0018. A method for fractionating mAbs into sub-pools, comprising:introducing an equilibration buffer to a column followed by a mAb solution; loading the mAb solution into a chromatography column;optionally washing the chromatography column;introducing an elution buffer into the chromatography column; starting a step pH gradient elution at 40 percent buffer B and decreasing to 10 percent buffer B, wherein buffer B operates in a neutral pH range;collecting a plurality of sub-pools of mAb with specific proteoforms at a given step pH value, wherein each sub-pool is enriched with different proteoforms of mAb;optionally characterizing the plurality of sub-pools;optionally reconstituting the plurality of sub-pools into a single pool comprising two or more proteoforms; andoptionally filtering the single pool.

9. The method of claim 8, wherein each sub-pool(s) of mAbs is collected such that as many sub-pools as there are different elution steps are collected at 37% buffer B, 33% buffer B, 30% buffer B, etc., to 10% buffer B, for collecting a plurality of sub-pools, wherein each sub-pool is enriched with different proteoforms of mAbs.

10. The method of claim 8 wherein the characterization step evaluates sub-pools for uniformity in concentration, pH, and conductivity.11.The method of claim 8, where the bioanalytical characterization(s) characterizes at least one of relative values for hydrophobicity, diffusion interaction parameter (ko), capillary isoelectric focusing (clEF), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC) and glycan profiles.

12. The method of claim 8, wherein the buffer B comprises tris(hydroxymethyl)aminomethane.

13. The method of claim 8, wherein the buffer B comprises acetic acid and sodium acetate.

14. The method of claim 8, wherein a resin within the chromatography column is a cationic exchange resin.

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