Method for purifying biomixtures

Abstract

Provided is a chromatographic method for separating components of a biomaterial mixture. The method employs displacement chromatography in the presence of a displacement enhancer to separate components of a biomaterial mixture comprising a high molecular weight biomaterial impurity and a target biomaterial.

Description

IGM Ref. No. 008-075 WO 1METHOD FOR PURIFYING BIOMIXTURESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 700,097, filed 27 September 2024 and 63 / 750,200, filed 27 January 2025, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Research and development of therapeutic proteins and other therapeutic macromolecules has grown significantly in recent years. Recombinant protein therapeutics have been developed to treat a variety of indications including, for example, cancer, autoimmune diseases, inflammation, and genetic disorders. Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases. See, e.g., U.S. Patent Nos. 9,951,134 and 9,938,347, and PCT Publication Nos. WO 2016 / 141303, WO 2016 / 154593, WO 2016 / 168758, WO 2017 / 059387, WO 2017 059380, WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, WO 2018 / 187702, and WO 2019 / 169314A1, the contents of which are incorporated herein by reference in their entireties.

[0003] Recombinant proteins for such applications are generally produced in living cells or organisms, including, for example, bacteria, mammalian cells, yeast, insect cells, transgenic plants, and transgenic animals. Cell-based expression typically involves suspension of cells in a cultivation medium or broth that contain salts, sugars, proteins and other various nutrients supporting growth of the particular cells. The desired product can be either secreted by the cells into the growth medium or retained within the cell body. The harvested cell medium is then processed to recover the desired product. The complexity of such biological mixtures, including differences in cell line, species origin, cell culture conditions, medium content, diversity of target molecule structures such as, for example, multimeric antibodies, and the like, present bioprocessing challenges for the efficient and reproducible recovery of purified recombinant proteins or other biomolecules.IGM Ref. No. 008-075 WO 1

[0004] Typically, multiple downstream processing steps are employed to provide high purity target biomolecules, such as, for example, centrifugation, cell disruption, molecular sieving, viral inactivation, precipitation, chromatography, filtration, etc. Challenges related to the bioprocessing of recombinant proteins or other biomolecules include, for example, large numbers of processing steps, low productivity, low recovery yields, and unacceptable impurity levels in the recovered product, among others. Particular challenges in protein purification include, for example, removal of protein aggregates and / or host-cell proteins (HCPs), while achieving high recoveries of intact product. There remains a need for improved and efficient methods for providing highly pure biomolecules, such as, for example, antibodies; such methods can, for example, provide increased throughput, a reduction in the number of processing steps, improved removal of impurities, such as, for example, high molecular weight impurities, and so forth, while additionally providing cost and / or time savings. It is believed that the methods provided herein meet one or more of the foregoing needs.SUMMARY

[0005] Provided herein is a method, i.e., a chromatographic method, for separating components of a biomaterial mixture comprising a high molecular weight biomaterial impurity (HMWI) and a target biomaterial having a lower molecular weight than the HMWI. Generally, the method comprises (a) applying an aqueous loading solution comprising the biomaterial mixture, a salt, and a displacement enhancer, such as, for example, a non-naturally occurring, non-ionic, water-soluble polymer such as, for example, poly(ethylene glycol), to a chromatography medium, to thereby initially bind components of the biomaterial mixture to the chromatography medium, and (b) continuously applying the aqueous loading solution to the chromatography medium to effect displacement of the target biomaterial from the chromatography medium, thereby providing an eluate comprising the target biomaterial at a higher purity relative to that of the loading solution.

[0006] In some embodiments, the method comprises (a) applying an aqueous loading solution comprising the biomaterial mixture, a salt, and 0.1% (w / v) to 10% (w / v) of a displacement enhancer, such as, for example, a non-naturally occurring, non-ionic, water-soluble polymer having an average molecular weight from about 300 to about 100,000 daltons, to a chromatography medium, to thereby initially bind components ofIGM Ref. No. 008-075 WO 1 the biomaterial mixture to the chromatography medium, and (b) continuously applying the loading solution to the chromatography medium to effect displacement of the target biomaterial from the chromatography medium, thereby providing an eluate comprising the target biomaterial at a higher purity relative to that of the loading solution.

[0007] In one or more embodiments of the method, step (b) is carried out in the absence of an elution gradient.

[0008] In some embodiments, the biomaterial mixture comprised in the loading solution contains from about 20 percent to about 1 percent HMWI of total biomaterial in the mixture. In some other embodiments, the biomaterial mixture comprised in the loading solution contains from about 18 percent to about 3 percent HMWI of total biomaterial in the mixture. In some further embodiments, the biomaterial mixture comprised in the loading solution contains from about 15 percent to about 7 percent HMWI of total biomaterial in the mixture, inclusive of all ranges falling between members of each of the foregoing ranges.

[0009] In one or more embodiments, the biomaterial mixture comprises at least one or more of a protein, peptide, virus, liposome, and / or nucleic acid.

[0010] In some further embodiments, the HMWI comprises an aggregated protein, a hostcell protein, a virus, a nucleic acid, or a combination of one or more of the foregoing.

[0011] In some embodiments, the target biomaterial (also referred as a target molecule, target biomolecule, target protein, or simply, target, and these terms may be used interchangeably herein) is a protein and the HMWI comprises at least one of an aggregated protein, host-cell protein, virus or a combination of one or more of the foregoing. In some further embodiments, the target biomaterial is an antibody or an antibody fragment. In yet some more particular embodiments, the antibody is an IgA or an IgM antibody.

[0012] As described above, the chromatography method includes the use of a displacement enhancer such as a non-naturally occurring, non-ionic, water-soluble polymer. In some embodiments, the non-naturally occurring, non-ionic, water-soluble polymer is a poly(ethylene glycol) (PEG), a polyacrylamide (PAM), a polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or a copolymer or combination thereof. In some particular embodiments of the method, the non-naturally occurring, non-ionic, water- soluble polymer is a poly(ethylene glycol) (PEG).IGM Ref. No. 008-075 WO 1

[0013] In some further embodiments, the non-naturally occurring, non-ionic, water- soluble polymer has an average molecular weight between about 300 daltons and about 40,000 daltons. In some embodiments, the non-naturally occurring, non-ionic, water- soluble polymer has an average molecular weight between about 350 daltons and about 30,000 daltons. In some other embodiments, the non-naturally occurring, non-ionic, water-soluble polymer has an average molecular weight between about 350 daltons and about 20,000 daltons. In some other embodiments, the non-naturally occurring, non- ionic, water-soluble polymer has an average molecular weight between about 350 daltons, and about 10,000 daltons. In some other embodiments, the non-naturally occurring, non-ionic, water-soluble polymer has an average molecular weight between about 350 daltons and about 6,000 daltons. In yet some additional embodiments, the non- naturally occurring, non-ionic, water-soluble polymer has an average molecular weight between about 400 daltons and about 5,000 daltons.

[0014] In one or more embodiments of the method, the aqueous loading solution further comprises a buffer. Exemplary buffers include, for example, a phosphate buffer, histidine, imidazole, a Good’s buffer, or a combination of one or more of the foregoing. Good’s buffers that can be comprised in the loading solution include, for example, one or more of the following: MES buffer, bis-tris buffer, ADA buffer, ACES buffer, PIPES buffer, MOPSO buffer, bis-6-tris propane buffer, BES buffer, MOPS buffer, TES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, TAPSO buffer, HEPPSO buffer, POPSO buffer, HEPPS buffer, tricine buffer, Gly-Gly buffer, bicine buffer, HEPBS buffer, TAPS buffer, AMPD buffer, TABS buffer, AMPSO buffer, CHES buffer, CAPSO buffer, AMP buffer, CAPS buffer, and CABS buffer.

[0015] In some embodiments, the pH of the aqueous loading solution ranges from about 5.0 to about 8.6. In some further embodiments, the aqueous loading solution comprises from about 0.10 percent to about 10 percent (e.g., w / v) of the non-naturally occurring, non-ionic, water-soluble polymer. In some other embodiments, the aqueous loading solution comprises from about 0.50 percent to about 5.0 percent (e.g., w / v) of the non- naturally occurring, non-ionic, water-soluble polymer.

[0016] In yet some additional embodiments, the salt comprised in the loading solution is sodium chloride, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, tris chloride or a combination of one or more of the foregoing. In some otherIGM Ref. No. 008-075 WO 1 embodiments, the salt comprised in the loading solution is a salt form of a buffer or buffer component.

[0017] In some embodiments, the loading solution comprises a salt concentration from about 75 mM to about 500 mM. In some other embodiments, the loading solution comprises a salt concentration from about 75 mM to about 250 mM.

[0018] In one or more embodiments of the method, the loading solution is applied at a loading density from about 15 mg / mL to about 350 mg / mL target biomaterial. In some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 300 mg / mL target biomaterial. In yet some other embodiments, the loading solution is applied at a loading density from about 25 mg / mL to about 200 mg / mL target biomaterial. In some further embodiments, the loading solution is applied at a loading density from about 25 mg / mL to about 100 mg / mL target biomaterial. In yet some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 80 mg / mL target biomaterial. In yet some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 50 mg / mL target biomaterial.

[0019] In one or more additional embodiments of the method, the loading solution is applied at a loading density from about 15 mg / mL to about 350 mg / mL of the biomaterial mixture. In some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 300 mg / mL of the biomaterial mixture. In yet some other embodiments, the loading solution is applied at a loading density from about 25 mg / mL to about 200 mg / mL of the biomaterial mixture. In some further embodiments, the loading solution is applied at a loading density from about 25 mg / mL to about 100 mg / mL of the biomaterial mixture. In yet some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 80 mg / mL of the biomaterial mixture. In yet some other embodiments, the loading solution is applied at a loading density from about 20 mg / mL to about 50 mg / mL of the biomaterial mixture.

[0020] In some embodiments of the method, the chromatography medium is an ion exchange medium, an affinity medium, for example, a metal affinity medium, a mixedmode medium, or a reverse phase medium. In yet some other embodiments, the chromatography medium is an ion exchange medium, an affinity medium, or a mixedmode medium. In yet some more particular embodiments, the chromatography mediumIGM Ref. No. 008-075 WO 1 is an ion exchange medium, such as, for example, an anion exchange medium or a cation exchange medium. In some additional embodiments, the chromatography medium is housed in a column. In yet some other embodiments, the chromatography medium is comprised in / on a filter.

[0021] In one or more embodiments of the method, the HMWI comprises a host cell protein, and the host cell protein content of the loading solution does not exceed 50,000 parts per million, or does not exceed 10,000 parts per million, or does not exceed 4,000 parts per million, with a lower range of, for example, about 10 parts per million.

[0022] In some further embodiments of the method, the target biomaterial is a multimeric binding molecule. Exemplary multimeric binding molecules include, for example, a multimeric antibody comprising two, four, five, or six bivalent binding units, wherein each binding unit comprises two heavy chains each comprising at least the heavy chain variable region of a binding domain and an IgA heavy chain constant region or multimerizing fragment or variant thereof, or an IgM heavy chain constant region or multimerizing fragment or variant thereof. In some particular embodiments of the method, the target biomaterial is a pentameric antibody. In yet some further embodiments, the target biomaterial is a hexameric antibody. In yet some other embodiments, the antibody is a tetrameric antibody. Exemplary multimeric antibodies are described in greater detail in the sections which follow.

[0023] In some additional embodiments, the method further comprises (c) recovering the target biomaterial displaced from the chromatography medium as a recovered target biomaterial.

[0024] In one or more further embodiments, the method is effective to remove 65 percent or more, 70 percent or more, 75 percent or more, 80 percent or more, or 90 percent or more HMWI from the biomaterial mixture in step (a).

[0025] In some embodiments of the method comprising step (c), the recovered target biomaterial contains less than 5 percent, less than 4 percent, less than 3 percent, or less than 2 percent HMWI. In some additional embodiments, the recovered target biomaterial contains no more than 0.25 percent HMWI.

[0026] In some embodiments of the method, the HMWI comprises a virus, and the method results in from 3 to 7 logs of virus clearance or from 4 to 7 logs of virus clearance from the biomaterial mixture.IGM Ref. No. 008-075 WO 1

[0027] In one or more further embodiments of the method, the biomaterial mixture has undergone one or more pre-purification steps prior to step (a). In some embodiments, the one or more pre-purification steps include a flow-through purification step. In some further embodiments, the one or more pre-purification steps include a capture step and / or one or more polishing steps. In yet another embodiment, following the one or more prepurification steps, the method further comprises flushing the chromatography medium with an aqueous buffer solution.

[0028] In some embodiments, the method further comprises, prior to applying step (a), or, as applicable, following the one or more pre-purification steps, washing the chromatography medium with an aqueous equilibration buffer having the same components and concentrations of components as the aqueous loading solution absent the biomaterial mixture.

[0029] In some particular embodiments of the method, the chromatography medium is an anion exchange medium, the loading solution comprises sodium chloride at a concentration ranging from about 75 mM to about 400 mM, sodium phosphate at a concentration ranging from about 1 mM to about 25 mM, and from about 0.5 to about 7 percent (w / v) poly(ethylene glycol) having an average molecular weight greater than about 300 daltons, e.g., from about 1000 to about 10,000 daltons.

[0030] In some embodiments of the method, the recovered target biomaterial contains less than 4 percent HMWI, less than 3 percent HMWI, or less than 2 percent HMWI.

[0031] In one or more particular embodiments of the method, the target biomaterial is imvotamab and the HMWI comprises imvotamab aggregates.

[0032] In one or more particular embodiments of the method, the target biomaterial is aplitabart and the HMWI comprises aplitabart aggregates.

[0033] In one or more additional embodiments, the method further comprises (d) isolating the recovered target material.

[0034] These and additional embodiments of the method are described in greater detail in the sections that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 illustrates the effect of increasing poly(ethylene glycol) (PEG) polymer size (1000, 1500, 3000, 4600 daltons) on separation of an exemplary biomaterial mixture containing a target biomaterial, the IgM antibody, imvotamab, during bind and eluteIGM Ref. No. 008-075 WO 1 chromatography using a ceramic hydroxyapatite chromatography column, CHT™-II (Bio-Rad, 0.8 cm diameter, 10 cm bed height, 5 mL total volume) in a buffer containing 5% (v / v) PEG. More particularly, FIG. 1 provides an overlay of different chromatograms for runs carried out with PEGs of various sizes.

[0036] FIG. 2 presents a non-comprehensive schematic of optional, additional purification steps that can be used in combination with the displacement chromatography method (also sometimes referred to herein as “polymer-assisted displacement chromatography”) of the present disclosure.

[0037] FIGs. 3A-D display the time-course of an illustrative chromatographic purification using a displacement method as described in the present disclosure, and as described in detail in Example 1. FIG. 3A is a processing chromatogram illustrating the complete chromatography run, where the X axis shows both collected fractions (top horizontal axis) and volume in milliliters (bottom horizontal axis) and the Y axis is mAU FIGs. 3B, 3C, and 3D are size exclusion chromatographs, where the X axis is retention time, and the peaks correspond to the molecular weight of protein species eluting from the column. FIG. 3B illustrates a peak corresponding to unaggregated (target) antibody present in fraction 1 A3, collected during chromatographic medium saturation (box with dotted line in FIG. 3A) during which time the protein in the sample saturates (i.e., binds to) sites on the chromatography medium. In some instances, a target biomaterial is referred to herein as a target biomolecule or target molecule, and these terms can be used interchangeably. FIG. 3C illustrates peaks corresponding to aggregated and non-aggregated antibody present in fractions over the course of loading and washout (box with dashed line in FIG. 3A). FIG. 3D illustrates peaks corresponding to aggregated and unaggregated antibody in the strip fraction (box with dashed and dotted line in FIG. 3A).

[0038] FIGs. 4A-C provide chromatographic analyses (i.e., chromatograms) of samples collected at three different time points over the course of an exemplary displacement chromatography purification of an imvotamab preparation containing 130 mg of total protein as described in detail in Example 2. FIG. 4A shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates (HMWI) in the unpurified preparation. FIG. 4B shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates in the purified preparation. FIG. 4C shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates in the mix ofIGM Ref. No. 008-075 WO 1 proteins that were stripped from the filter following displacement chromatographic purification.

[0039] FIGs. 5A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatography purification of an imvotamab preparation containing 14 g of total protein as described in detail in Example 2. FIG. 5A shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates (HMWI) in the unpurified preparation. FIG. 5B shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates in the purified preparation. FIG. 5C shows the relative prevalence of imvotamab (target biomolecule) and imvotamab aggregates in the mixture of proteins that were stripped from the filter following displacement chromatographic purification.

[0040] FIGs. 6A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a 50 mg total antibody sample containing a bispecific, pentameric IgM antibody with ten binding domains that bind to CD38 and a single anti-CD3 binding domain on the J chain as described in Example 3. The IgM antibody in the sample preparation has a lower molecular mass than imvotamab (molecular weight of about 976,862 daltons). FIG. 6A shows the relative prevalence of target biomolecule and aggregates in the unpurified sample preparation. FIG. 6B shows the relative prevalence of target biomolecule and aggregates in the preparation purified in accordance with the methods provided herein. FIG. 6C shows the relative prevalence of target biomolecule and aggregate in the mixture of proteins that were stripped from the filter following displacement chromatographic purification.

[0041] FIGs. 7A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a 310 mg total antibody sample containing a bispecific, pentameric IgM antibody with ten binding domains that bind to CD38 and a single anti-CD3 binding domain on the J chain as described in Example 3. FIG. 7A shows the relative prevalence of target biomolecule and aggregate in the unpurified sample preparation. FIG. 7B shows the relative prevalence of target biomolecule and aggregates in the preparation purified in accordance with the methods provided herein. FIG. 7C shows the relative prevalence of target biomolecule and aggregate in the mixture of proteins that were stripped from the filter following displacement chromatographic purification.IGM Ref. No. 008-075 WO 1

[0042] FIGs. 8A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a 100 mg total antibody sample containing an anti-SARS-CoV-2 pentameric IgM antibody having a lower molecular mass than imvotamab as described in Example 4. FIG. 8A shows the relative prevalence of target biomolecule and aggregates in the unpurified preparation. FIG. 8B shows the relative prevalence of target biomolecule and aggregate in the preparation purified in accordance with the methods provided herein. FIG. 8C shows the relative prevalence of target biomolecule and aggregates in the mixture of proteins that were stripped from the filter following displacement chromatography purification.

[0043] FIGs. 9A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a 150 mg total antibody sample containing an anti-SARS-CoV2 pentameric IgM antibody having a lower molecular mass than imvotamab as described in Example 4. FIG. 9A shows the relative prevalence of target biomolecule and aggregates in the unpurified preparation. FIG. 9B shows the relative prevalence of target biomolecule and aggregates in the preparation purified in accordance with the methods provided herein. FIG. 9C shows the relative prevalence of target biomolecule and aggregates in the mixture of proteins that were stripped from the filter following displacement chromatography purification.

[0044] FIGs. 10A-C provide chromatographic analyses corresponding to three different displacement chromatography purification runs of preparations of an exemplary IgM antibody, imvotamab, using a retrovirus as a displacer molecule as described in Examples 5 and 6. For each of FIGs. 10A-C, the plot shows (from left to right) the relative prevalence of target biomolecule and aggregates (HMWI) in the unpurified preparation, the relative prevalence of target biomolecule and aggregates in the preparation purified in accordance with the methods provided herein, and the relative prevalence of target biomolecule and aggregates in the mixture of proteins that were stripped from the filter following displacement chromatography purification. For the starting preparation corresponding to FIG. 10A, no retrovirus was added to the preparation prior to displacement chromatography. For the starting preparation corresponding to FIG. 10B, a small quantity of retrovirus was added to the preparationIGM Ref. No. 008-075 WO 1 prior to purification. For the starting preparation corresponding to FIG. 10C, a larger quantity of retrovirus was added to the preparation prior to purification.

[0045] FIGs. 11A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a 274 mg total protein sample containing both non-aggregate and aggregates of a CD123 x CD3 bispecific, pentameric IgM antibody with ten binding domains that bind to CD 123 and a single CD3s binding domain on the J chain as described in Example 7. FIG. 11A shows the relative prevalence of target biomolecule and aggregates in the unpurified preparation. FIG. 11B shows the relative prevalence of target biomolecule and aggregates in the preparation purified in accordance with the methods provided herein. FIG. 11C shows the relative prevalence of target biomolecule and aggregates in the mixture of proteins that were stripped from the filter following displacement chromatography purification.

[0046] FIGs. 12A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a sample (Sample 1) containing both non-aggregate and aggregates of an anti-DR5 pentameric IgM antibody as described in Example 8. FIG. 12A provides a chromatographic analysis of Sample 1 prior to purification, where aggregate amounted to 6.74% of the total protein in the sample. FIG. 12B provides a chromatographic analysis of the purified preparation following displacement chromatography, illustrating a reduction in aggregate to 2.32% of the total protein content. Following stripping of the filter following chromatographic purification (FIG. 12C), chromatographic analysis of the strip fraction revealed that 29.72% of the total protein was aggregate, again illustrating that the aggregate preferentially accumulated on the filter.

[0047] FIGs. 13A-C provide chromatographic analyses of samples collected at three different time points over the course of an exemplary displacement chromatographic purification of a sample (Sample 2) containing both non-aggregate and aggregates of an anti-DR5 pentameric IgM antibody as described in Example 8. FIG. 13A provides a chromatographic analysis of Sample 2 prior to purification, where aggregate amounted to 2.90% of the total protein in the sample. FIG. 13B provides a chromatographic analysis of the purified preparation following displacement chromatography, illustrating a reduction in aggregate amount to 1.20% of the total protein content. Following stripping of the filter following purification (FIG. 13C), chromatographic analysis revealed thatIGM Ref. No. 008-075 WO 117.13% of the total protein in the strip fraction was aggregate, illustrating that the aggregate preferentially accumulated on the filter.

[0048] FIGs. 14A-E display the chromatographic purification of a biomixture comprising an exemplary anti-DR5 pentameric antibody, aplitabart (IGM-8444), using polymer- assisted displacement chromatography with a ceramic hydroxyapatite chromatography medium, as described in Example 10. FIG. 14A illustrates the complete chromatography run, where the top horizontal axis represents collected fractions, the bottom horizontal axis represents volume in milliliters, and boxed regions correspond to samples collected and analyzed over the course of the chromatography run. FIGs. 14B-E are size exclusion chromatographs, where the X axis is retention time and the peaks correspond to the molecular weight of protein species eluting from the column and the Y axis is mAU. FIG. 14B provides a chromatographic analysis of the biomaterial that is loaded onto the column. FIG. 14C provides an analysis of the purified preparation collected at the beginning of the process. FIG. 14D provides a chromatographic analysis of the purified preparation at the end of sample loading. FIG. 14E provides a chromatographic analysis of the purified preparation at the end of displacement chromatography.DETAILED DESCRIPTIONDefinitions

[0049] As used herein, the term “a” or “an” in reference to an entity refers to one or more of that entity. For example, “a high molecular weight biomaterial impurity” is understood to represent one or more high molecular weight biomaterial impurities. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0050] The term, “and / or” where used herein is to be taken as specific disclosure of each of the specified features or components which follow such term with or without the other. That is, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).IGM Ref. No. 008-075 WO 1

[0051] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2d ed., 2006, Oxford University Press; Biopharmaceutical Manufacturing, Volumes 1 (2021) and 2 (2022), Niazi, S., Lokesh, S., IOP Publishing Ltd, and Biopharmaceutical Manufacturing: Principles, Processes and Practices, Gilleskie, F., Rutter, C., McCuen, B., De Gruyter, 1stedition (2021), provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0052] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be understood by reference to the specification as a whole. Accordingly, the terms defined herein are more fully defined by reference to the specification in its entirety.

[0053] The term, “chromatography”, generally refers to a process of separating components of a mixture by distributing the components between a stationary phase and a mobile phase. The stationary phase comprises one or more immobilized species that interact chemically and / or physically with the molecules to be separated; the mobile phase, e.g., in liquid chromatography, comprises one or more solvents that carry components of a mixture to be separated through the stationary phase.

[0054] In the methods provided herein, the term, “chromatography medium” refers to a stationary phase; the terms “chromatography medium”, “stationary phase”, “solid phase” and “solid support” are used herein interchangeably. The stationary phase can, for example, be comprised in or on a column, cartridge, filter, membrane, or other suitable container or supporting surface. By way of non-limiting examples, both resin columns and filters are suitable chromatography media for use in the methods presently disclosed.

[0055] “Load density” or “loading density” as used herein refers to the amount of a composition, e.g., a biomaterial mixture, or a component of a biomaterial mixture such as a target biomaterial (e.g., a target protein), placed in contact with a volume of a chromatography medium such as, for example, a chromatography column having a particular volume, or in the case of a filter, its chemically active bed volume. CommonIGM Ref. No. 008-075 WO 1 units of measurement include but are not limited to, for example, milligrams per milliliter, or grams per liter.

[0056] A “single mode chromatography medium” conveys a chromatographic medium with a single chemical interaction / adsorption mechanism, such as, for example, cation exchange, anion exchange, etc.

[0057] A “mixed mode chromatography medium” is a medium (i.e., stationary phase) comprising immobilized ligands which interact with molecules to be separated through more than one type of interaction mechanism, such as, for example, cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi- pi bonding, and metal (e.g., calcium) affinity.

[0058] “Bind-elute” chromatography is a chromatographic approach in which a target biomaterial is loaded (i.e., adsorbed) onto a chromatography medium, typically followed by washing the medium to remove unbound species from the stationary phase, followed by desorption of the target biomaterial by changing the conditions of the mobile phase (e.g., ionic strength or pH) to elute the desired target biomaterial from the chromatography medium.

[0059] “Flow-through” chromatography (FTC) generally refers to a chromatographic method in which the target biomaterial is eluted without adsorption / interaction with the chromatography medium, while one or more impurities or contaminants are immobilized thereon.

[0060] The term, “biomaterial”, as used herein refers to any substance that is or can be derived from or produced by a biological organism (e.g., a plant, animal, bacteria, fungi, etc.), including, for example, proteins, peptides, polynucleotides, or other macromolecules in non-aggregated or aggregated form, viruses, and the like.

[0061] A “high molecular weight biomaterial impurity” or “HMWI” as used herein refers to an impurity or in some cases, a contaminant, present in a biomaterial mixture, where the HMWI, such as for example, an aggregate of a target protein, a host cell protein, and / or a virus, among other things, has a higher molecular weight than a target biomaterial to be separated. In the context of biological manufacturing, an impurity is a substance that is either process- or product-related, e.g., is present in the raw materials or formed during the manufacturing process other than the target molecule, while a contaminant is a material that is foreign to the process, such as, e.g., microorganisms such as bacterial or viral pathogens.IGM Ref. No. 008-075 WO 1

[0062] An “aggregate” in the context of a biomaterial mixture, refers to an agglomeration of multiple (e.g., of two or more, e.g., 5, 20, 50, etc.) smaller biomaterial units, such as, for example, protein aggregates, cell aggregates, and / or polynucleotide aggregates, with the understanding that in the case of aggregated protein, in the context of a multimeric binding molecule: a single IgA dimer is not an “aggregate” as the term is used herein; a single IgM pentamer is not an “aggregate” as the term is used herein; and a single IgM hexamer is not an “aggregate” as the term is used herein. Nevertheless, IgA dimers, IgM pentamers, and IgM hexamers can associate together with themselves, or with other molecules (including other antibodies) to form “aggregates.” Aggregates can result from covalent interactions, non-covalent interactions, or a combination of both.

[0063] A “target biomaterial” as used herein refers to a biomaterial component comprised in a biomaterial mixture to be separated in accordance with the polymer-assisted displacement chromatography methods provided herein; the target biomaterial has a lower molecular weight than the HMWI to be separated from the target biomaterial. Target biomaterials include, for example, peptides, proteins, polynucleotides, and other macromolecules in non-aggregated form. In some instances herein, a target biomaterial is referred to as “target”, “target biomolecule”, “target protein”, or “target molecule”, and these terms are used interchangeably herein.

[0064] A “non-naturally occurring” polymer is one that, in its entirety, is not found in nature. Non-naturally occurring polymers are also referred to as synthetic polymers.

[0065] A “water-soluble” polymer for use in the methods described herein is a polymer that is soluble in water or in aqueous solution at a concentration and under run conditions used in a chromatographic separation, e.g., displacement chromatography, as provided herein.

[0066] “Poly(ethylene glycol)” or “PEG” refers to a polymer comprising the following repeat unit, -(CH2CH2O)n-, where n is an integer of about 6 or greater. Poly(ethylene glycol) for use in the methods disclosed herein, e.g., as a displacement enhancer, possesses chemically inert end capping groups.

[0067] A “chemically inert” substance or functional group, such as a terminal end-capping group on a polymer such as PEG, is one that is not chemically reactive under standard conditions of use, for example, under typical ambient conditions during a chromatographic separation, such as the polymer-assisted displacement chromatography method disclosed herein, and is not chemically reactive in any pre- or post-processingIGM Ref. No. 008-075 WO 1 steps comprising the chemically inert, i.e., non-reactive, substance or functional group. Non-limiting examples of chemically inert or non-reactive substances for use in the polymer-assisted displacement chromatographic methods provided herein include poly(ethylene glycol) having terminal hydroxyl groups (sometimes referred to as PEG diol), and methoxy poly(ethylene glycol), also referred to as mPEG.

[0068] Molecular weight, in the context of a non-naturally occurring, non-ionic, water- soluble polymer, such as, for example, poly(ethylene glycol), is typically provided as an average molecular weight since synthetic polymers generally possess a distribution of molecular weights. The average molecular weight of a polymer can be determined using a variety of techniques, such as, for example, gel permeation chromatography, lightscattering measurements, viscosity measurements, etc. Polymer molecular weight is typically expressed as either a number average or a weight average. The average molecular weight of a non-naturally occurring, non-ionic, water-soluble polymer such as poly(ethylene glycol) is typically provided by the vendor, along with its basis for determination. In some instances, the average molecule weight of a commercially available water-soluble polymer such as PEG is indicated by a hyphenated suffix, e.g., PEG-6000, where “6000” refers to an average molecular weight of about 6,000 daltons, or PEG-4600, where “4600” refers to an average molecular weight of about 4600 daltons. In the absence of information to the contrary, e.g., by a vendor, it can be assumed that polymer average molecular weights are expressed as weight averages.

[0069] By “substantially” or “essentially” is meant nearly totally or completely, for instance, 95% or greater of a given quantity or feature, up to and including one hundred percent of the given quantity or feature.

[0070] As used herein, the terms “about” or “approximately” refer to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art. As used herein, the foregoing terms encompass a range within plus or minus five percent of a given value.

[0071] As used herein, “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). “Polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,”“amino acid chain,” or any other term used to refer to a chain or chains of two or moreIGM Ref. No. 008-075 WO 1 amino acids are included within the definition of "polypeptide,” and “polypeptide” can be used instead of any of these terms. “Polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0072] A polypeptide as described herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as “folded.” Polypeptides which do not possess a defined three-dimensional structure but rather can adopt many different conformations are referred to as “unfolded.”

[0073] An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0074] The terms “fragment,” “variant,” “derivative,” and “analog” as used herein include any polypeptides that retain at least some of the properties of the corresponding parent antibody or polypeptide from which the fragment, variant, derivative, or analog is derived or in reference to, for example, specific binding to an antigen. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments. Variants of, e.g., a polypeptide, include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. For example, variants can be non-naturally occurring. Non-naturally occurring variants can be produced using art- known mutagenesis techniques. Variant polypeptides can comprise conservative or non-IGM Ref. No. 008-075 WO 1 conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. As used herein a “derivative” of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those polypeptides that contain one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5- hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0075] “Polynucleotide” encompasses a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0076] An “isolated” nucleic acid or polynucleotide conveys any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.” Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0077] “Antibody” and “immunoglobulin” are used interchangeably herein. An antibody includes at least the variable domain of a heavy chain (e.g., from a camelid species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulinIGM Ref. No. 008-075 WO 1 structures in vertebrate systems are relatively well understood. See, e.g., Greenfield, E.A. (ed.), Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 2013). Unless otherwise stated, “antibody” encompasses anything ranging from a small antigen-binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, a dimeric or tetrameric IgA antibody that includes four or eight complete heavy chains and four or eight complete light chains and includes a J chain and / or a secretory component, or a pentameric or hexameric IgM antibody or IgM-like antibody, that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J chain or functional fragment or variant thereof.

[0078] “Immunoglobulin” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, a, 5, s) with some subclasses among them (e.g., yl-y4 or al-ot.2)). It is the nature of this chain that determines the “isotype” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgGi, IgG?, IgG?, IgG4, IgAi, IgA?, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan and, accordingly, are within the scope of this disclosure.

[0079] As used herein, “binding domain” or “antigen-binding domain” can be used interchangeably and refers to a region of a binding molecule, e.g., an antibody or antibody-like molecule, that is necessary and sufficient to bind specifically to a target, e.g., an epitope, a polypeptide, a cell, or an organ. For example, an “Fv,” e.g., a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is a “binding domain.” Other antigenbinding domains include, without limitation, a single domain heavy chain variable region (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold. A “binding molecule,” e.g., an “antibody” as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more “antigen-binding domains.”

[0080] “Binding unit” is used herein to refer to the portion of a binding molecule, e.g., an antibody or antibody-like molecule, that corresponds to a standard immunoglobulinIGM Ref. No. 008-075 WO 1 structure, e.g. an “H2L2” immunoglobulin structure of two heavy chains and two light chains, or, for certain heavy chain-only antibodies an “H2” immunoglobulin structure. In certain embodiments, e.g., where the binding molecule is a bivalent IgG antibody, the terms “binding molecule” and “binding unit” are equivalent. Such a binding molecule is also referred to herein as “monomeric.” In other embodiments, e.g., where the binding molecule is a “multimeric binding molecule,” e.g., a dimeric or tetrameric IgA antibody or IgA-like antibody or a pentameric or hexameric IgM antibody or IgM-like antibody, the binding molecule comprises two or more “binding units” — two in the case of an IgA dimer, four in the case of an IgA tetramer, five in the case of an IgM pentamer, or six in the case of an IgM hexamer. A binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” as defined above. As used herein, certain examples of a binding molecule provided in this disclosure are “dimeric,” and include two bivalent binding units that include IgA constant regions or multimerizing fragments thereof. Certain examples of a binding molecule provided in this disclosure are “pentameric” or “hexameric,” and include five or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof. A binding molecule, e.g., an antibody or antibody -like molecule comprising two or more, e.g., two, five, or six binding units, is referred to herein as “multimeric.”

[0081] “J chain” as used herein refers to the joining chain of an IgM or IgA antibody of any animal species or any functional fragment and / or variant thereof. As persons of ordinary skill in the art will recognize, “a functional fragment” or “a functional variant” of a J chain includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody or can associate with IgA heavy chain constant regions to form a dimeric IgA antibody. Exemplary modified J chains can be found, e.g., in U.S. Patent Nos. 9,951,134, 10,400,038, 10,618,978, and 11,639,389.

[0082] The terms “valency,” “bivalent,” “multivalent” and grammatical equivalents refer to the number of binding domains, e.g., antigen-binding domains in given binding molecule, e.g., antibody or antibody -like molecule, or in a given binding unit. As such, “bivalent,” “tetravalent,” and “hexavalent” in reference to a given binding molecule, e.g., an IgM antibody or an IgM-like antibody denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding domains, respectively.IGM Ref. No. 008-075 WO 1A typical IgM antibody or IgM-like antibody where each binding unit is bivalent, can have 10 or 12 valencies. A bivalent or multivalent binding molecule, e.g., antibody or antibody -like molecule, can be monospecific, z.e., all of the antigen-binding domains are the same, or can be bispecific or multispecific, z.e., where two or more antigen-binding domains are different, e.g., bind to different epitopes on the same antigen, or bind to entirely different antigens.

[0083] Both the light and heavy chains of antibodies or antibody-like molecules are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant region domains of the light chain (CL) and the heavy chain (e.g., CHI, hinge, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N- terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 (or CH4, e.g., in the case of IgM) and CL domains comprise the carboxyterminus of the heavy and light chain, respectively.

[0084] A “full length IgM antibody heavy chain” is a polypeptide that includes, in an N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4), and can further include an IgM tail-piece.

[0085] A “full length IgA antibody heavy chain” is a polypeptide that includes, in N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CAI or Cal), an IgA hinge region, an antibody heavy chain constant domain 2 (CA2 or Ca2), and an antibody heavy chain constant domain 3 (CA3 or Ca3), and can further include an IgA tail-piece.

[0086] As indicated above, variable region(s) allow a binding molecule, e.g., an antibody or antibody-like molecule, to recognize selectively and bind specifically to epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs) of a binding molecule, e.g., an antibody or antibody -likeIGM Ref. No. 008-075 WO 1 molecule, combine to form the antigen-binding domain. More precisely, an antigenbinding domain can be defined by three complementarity determining regions (CDRs) on each of the VH and VL chains. Certain antibodies form larger structures. For example, IgA can form a molecule that includes two or four H2L2 binding units and a J chain covalently connected via disulfide bonds, which can be further associated with a secretory component, and IgM can form a pentameric molecule that includes five H2L2 binding units and a J chain, or hexameric molecule that includes six H2L2 binding units, each covalently connected via disulfide bonds.

[0087] The six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, referred to as “framework” regions, show less inter- molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non- covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health & Human Services, (1983); and Chothia & Lesk (1987) J. Mol. Biol. 196:901-17, which are incorporated herein by reference in their entireties).

[0088] In the case where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. General Overview

[0089] The methods disclosed herein relate to downstream process design, namely, chromatographic purification of complex biomaterial mixtures using displacement chromatography. The manufacturing of complex biomolecules, such as, for example,IGM Ref. No. 008-075 WO 1 recombinant proteins, is frequently accompanied by formation of both product and process-related impurities, including high molecular weight impurities (HMWIs) such as, for example, protein aggregates, host-cell proteins, etc., which can be difficult to remove to pharmaceutically acceptable levels. Such impurities, if not effectively removed, can adversely impact the safety, bioactivity, and stability of a purified biomolecule (e.g., a drug substance) and its drug product formulation. In investigating chromatographic approaches for effectively removing high molecular weight impurities from a target biomolecule mixture, it has been discovered that addition of a displacement enhancer molecule (to be described in greater detail below) can be utilized during displacement chromatography to effectively and notably improve the purity of a recovered target biomolecule with respect to the removal of undesirable high molecular weight impurities.

[0090] Displacement chromatography (sometimes referred to as overload chromatography) is a chromatographic method in which a molecule with a high affinity for the chromatography medium under elution conditions effectively competes for binding sites and displaces another molecule having a lesser affinity for the chromatography medium from the medium in the displacement train, that is, during elution. See, for example, Heikaus, L., Schluter, H., Am Pharm Rev., 2014, 17(2), and McAtee, C.P., Displacement Chromatography of Proteins, Current Protocols in Protein Science, Chapter 8, Unit 8.9 (2010), Ed. Board,. John E. Corrigan, et al., for a general description of displacement chromatography of proteins. In arriving at the chromatographic purification methods disclosed herein, it was discovered that addition of a displacement enhancer to an aqueous loading solution comprising a target biomolecule and one or more high molecular weight impurities was effective to promote displacement of the target biomolecule from the chromatography medium with selective retainment of high molecular weight impurities on the chromatography medium over the target biomolecule, to thereby remove, to a large extent, such impurities from the biomaterial mixture. That is, in the presence of the displacement enhancer, intact, nonaggregated target biomolecules are out-competed and selectively displaced by the HMWIs which are then largely retained on the chromatography medium to provide an eluate that is enriched, often to a significant extent, in target molecule having a higher purity relative to that of the loading solution.IGM Ref. No. 008-075 WO 1Description of the Method

[0091] As disclosed above, the chromatographic methods disclosed herein utilize a displacement enhancer in a loading solution to facilitate displacement of a target biomaterial from a stationary phase following application of an aqueous loading solution containing a biomaterial mixture comprising the target biomaterial and a high molecular weight impurity (e.g., aggregated protein, host cell proteins, host cell DNA and RNA, viruses), a salt, and the displacement enhancer onto the stationary phase. The loading solution is then continuously applied to the chromatography medium to effect displacement of the target biomaterial therefrom, to thereby provide an eluate comprising the target biomaterial at a higher purity relative to that of the loading solution. It has been discovered that a particularly effective displacement enhancer is a non-naturally occurring, non-ionic, water-soluble polymer, such as, for example, poly(ethylene glycol); in some instances herein, the chromatographic separation method provided is referred to generally as polymer-assisted displacement chromatography.

[0092] More particularly, the method comprises continuously loading onto a chromatography medium, a biomaterial mixture comprising a target biomolecule (such as, e.g., an IgM or an IgA antibody) and one or more high molecular weight impurities (HMWI) comprised in an aqueous solution comprising a salt and a suitable amount (e.g., from about 0.1% to about 10% w / v) of a non-naturally occurring, non-ionic, water- soluble polymer such as, e.g., PEG (e.g., PEG-400, PEG-1500, PEG-3350, PEG-4000, PEG-4600, PEG-6000, PEG-8000, etc., having an average molecular weight from about 300 to about 100,000 daltons), until the chromatography medium is substantially saturated (and where the target biomaterial has a lower molecular weight than the HMWI). Upon saturation of the chromatography medium, and in the presence of the displacement enhancer, the HMWI begins to outcompete the target biomolecule for binding to the chromatography medium. As the biomaterial mixture moves through the medium, the HMWI (e.g., aggregates, host-cell protein, virus) displaces the target biomolecule from the chromatography medium. In other words, by calibrating the aqueous loading solution with displacement enhancer, i.e., the non-naturally occurring, non-ionic, water-soluble polymer, the chromatography method becomes a displacement chromatographic separation in which the HMWI acts as a displacer to the target biomolecule, such that eluate comprising the target biomolecule elutes from the chromatography medium as more of the impure biomaterial mixture passes through theIGM Ref. No. 008-075 WO 1 medium during continuous loading of the aqueous loading solution. In the method provided herein, occupation of available binding sites on the chromatography medium occurs before the target biomolecule is selectively displaced from the medium by the presence of large molecule impurities (i.e., molecular aggregates and the like). Generally, the aqueous loading solution is continuously applied to the chromatography medium until the chromatography medium becomes substantially saturated with the HMWI and a high percentage of the target biomaterial is displaced therefrom, to thereby recover a highly purified target biomolecule. In some embodiments, once all of the loading solution has been applied to the chromatography medium, an aqueous wash (chase) buffer is applied to the chromatography medium to push out remaining target molecule that remains (e.g., is loosely bound) thereto. The displacement chromatography method provided is highly efficient, and is distinct from traditional bind-elute and flow- through chromatography methods. The method is unique in that it utilizes, in the presence of the displacement enhancer, the high molecular weight biomaterial impurity(ies) to be removed as an agent to facilitate the chromatographic purification.

[0093] While in no way intending to be bound by theory or purported mechanism, the method utilizes the unique properties of non-naturally occurring, non-ionic, water- soluble polymers such as PEG as displacement enhancers, e.g., to alter the chemical potential of a target biomolecule. Polymers such as PEG can sterically exclude water to the hydration shell of a protein, such that in the presence of a chemically active chromatography medium, it is believed that preferential hydration allows the target biomolecule and the chromatography medium to share hydration water. See, e.g., Arakawa, T., Timasheff, S.N., Biochemistry (1985), 24, 6756-6762; Gagnon, P., J. of Immunological Methods, (2008), 336, 222-228; Schachman, L., Lauffer, A., (1949), J. Am. Chem. Soc., 71, 536-541. As increasing water is shared, binding of the target biomolecule to the medium becomes more energetically favorable, to thereby facilitate separation of components of complex biomaterial mixtures by size. This effect scales with both the hydrodynamic radius of the target biomolecule and the polymeric displacement enhancer, thereby facilitating separation by size. See, for example, FIG. 1. The presence of the polymeric displacement enhancer also enhances the binding of the HMWI to the stationary phase, such that following adsorption of the target biomolecule to the stationary phase via saturation of binding sites, the HMWI becomes the “displacer” molecule, outcompeting and displacing the target biomolecule from theIGM Ref. No. 008-075 WO 1 stationary phase, since the HMWI, having a larger size than the target biomolecule, gains affinity for the chromatography medium at a faster rate than the target. Generally, the larger (or greater the molecular weight of) a biomolecule, the more energy (solute) is required to remove such biomolecule from the chromatography medium. Thus, the method utilizes the presence of a high molecular weight impurity (or other high molecular weight molecule) in a target biomolecule-containing mixture, in the presence of a polymeric displacement enhancer as described, to facilitate effective removal of the HMWI and purification of the target biomolecule. The method is effective to remove both product (e.g., aggregated protein) and process (e.g., host cell protein)-related impurities, including clearance of viruses, to provide highly pure biomolecules, as illustrated in the supporting examples, and can be optimized for purifying any of a number of biomolecules by suitably adjusting process parameters, including the components of the mobile phase, as is well within the capabilities of the skilled artisan when taken with the instant disclosure. Various components and features of the method will now be described.

[0094] Stationary phase: Any of a number of stationary phases can be used in the method, depending upon the components of the biomaterial mixture. One illustrative stationary phase is, for example, an ion exchange medium, in which functional groups are attached to an insoluble matrix or supporting surface. The ion exchange medium can be, for example, an anion exchange (AEX) medium (comprising positively charged functional groups which can bind and / or exchange negatively charged ions), a cation exchange (CEX) medium (comprising negatively charged functional groups which can bind and / or exchange positively charged ions), or a mixed-mode medium, depending upon the target biomolecule to be purified. The chromatography medium can be comprised in or on a column, cartridge, filter, membrane, or other suitable container or supporting surface. The medium can comprise, for example, a strong or weak anion or cation exchanger, or a combination thereof. Functional groups comprised in an anion exchange medium include, for example, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), dimethylaminopropyl (DMAP), quaternized polyethyleneimine, trimethylaminoethyl (TMAE), and fully quaternized ammonium. Chromatography media suitable for use in the methods provided herein are commercially available from any of a number of vendors such as, e.g., Thermo Fisher Scientific, Inc., 3M, Sartorius AG, and Cytiva, to name a few. Illustrative AEX chromatography media include, e.g., POROS® anionIGM Ref. No. 008-075 WO 1 exchange resins (XQ, HQ 50, PI 50, D 50), 3M® EMPHAZE AEX Hybrid Purifier, SARTOBIND Q and MUSTANG QXT. POROS® anion exchange resins include a resin backbone of crosslinked poly(styrene divinylbenzene), and different surface chemistries. POROS® XQ resin contains quaternary amino groups; POROS® HQ 50 resin contains quaternary polyethyleneimine groups, POROS® PI 50 resin contains polyethylene imine groups, and POROS® D50 resin contains dimethylaminopropyl groups. 3M® EMPHAZE™ AEX Hybrid Purifier (also referred to simply as “EMPHAZE™”), also useful in the present methods, is a chromatographic purifier that contains a Q-functional anion exchange medium and a bioburden reduction membrane. The purifier contains three components including an anion exchange nonwoven, microporous membrane, and membrane support. EMPHAZE™ comprises an anion exchange medium of a quaternary ammonium functional polymer supported by a fine fiber nonwoven scaffold and is available in a variety of sizes including laboratory, pilot and production scale with products including BVO.3R, BV1R, BV8R, BV6OR, BV120R, BV360R, BV800R and BV5600R (see, e.g., 3M® EMPHAZE™ AEX Hybrid Purifier Product Information Sheet). Additional filtration media comprising an anion exchange chromatographic medium suitable for use in the present methods are described, e.g., in U.S. Patent No. 10,722,848. Anion exchange resins available from Sartorius include, for example, Q CERAMIC HYPERD® (a strong anion exchanger with quaternary amine groups), HyperCel STAR AX (a weak anion exchanger), DEAE Ceramic HYPERD®. Anion exchange resins available from Cytiva include resins from the Sepharose Fast Flow platform, e.g., ANX Sepharose Fast Flow (a weak ion exchanger) with tertiary amine groups attached to the base matrix Sepharose 4 Fast Flow, as well as Capto DEAE ion exchange resin (weak anion exchanger), Capto Q ImpRes resin (a strong anion exchanger), and DEAE Sephacel (a weak anion exchanger) as an example. Similarly, cationic exchange resins and mixed mode resins are readily available by the above-noted vendors in addition to many others. Cationic exchange resins typically include a sulfonic acid (strong cation exchanger) or carboxylic acid or phosphonic acid functional group (weak cationic exchangers), such as, e.g., sulfonate, sulfopropyl, carboxyl, or carboxymethyl. Examples of such commercially available resins include POROS® CEX, POROS® HS and XS strong cation exchange (SCX) resins (Thermo Fisher Scientific, Inc.), CAPTO™ S and SP Sepharose Fast Flow strong cation exchange resins (Cytiva), and mixed mode resins such as POROS® caprylate mixed mode cation exchange resinIGM Ref. No. 008-075 WO 1(Thermo Fisher Scientific, Inc.), CAPTO ™ adhere ImpRes (Cytiva) resin and CAPTO ™ MMC ImpRes resin (Cytiva). Another illustrative mixed mode stationary phase is ceramic hydroxyapatite, such as, for example, CHT™ ceramic hydroxyapatite (Bio-Rad Laboratories, Inc.), available as Type I and Type II. CHT is a mixed-mode support; it is a chemically pure form of hydroxyapatite that has been sintered at high temperatures; its functional groups include phosphate, calcium, and hydroxyl (Chromatography Bulletin 5667, 2013, Bio-Rad Laboratories, Inc.). Similar resins suitable for use in the methods provided herein are available from a number of different vendors in addition to those described above.

[0095] In certain embodiments, the stationary phase is packed in a column. Depending on the scale of the separation, the internal diameter of the column can range from, for example, 1 millimeter to 2 meters. The column can have, in some embodiments, an internal diameter of at least about or equal to about 3 millimeters, 4 millimeters, 5 millimeters, 6 millimeters, 7 millimeters, 8 millimeters, 9 millimeters, 10 millimeters, 20 millimeters, 50 millimeters, 75 millimeters, 10 centimeters, 50 centimeters, 75 centimeters, 1 meters, 1.25 meters, 1.50 meters, 1.75 meters, or 2 meters, or have an internal diameter falling within a range between any two of the foregoing values, and a bed height of at least about or equal to about 15 millimeters, 20 millimeters, 25 millimeters, 30 millimeters, 40 millimeters, 50 millimeters, 1 centimeter, 10 centimeters, 15 centimeters, 20 centimeters, 25 centimeters, or more, including having a bed height falling within a range of any two of the foregoing values. These dimensions can be useful for evaluating the effects of various chromatographic conditions on a particular antibody or protein to be separated from high molecular weight impurities. Other embodiments employ columns of various dimensions to support preparative chromatographic separations or commercial stage chromatographic purifications. Illustrative column diameters can range, for example, from less than 1 centimeter to greater than 1 meter, and column heights can range from, for example, less than 1 centimeter to greater than 30 centimeters, depending upon the scale of the chromatographic separation, the relative amounts and components of the biomaterial mixture, along with other factors. Appropriate column dimensions can be determined by the skilled artisan, based upon a consideration of factors related to the separation, and the method is not limited in this regard.IGM Ref. No. 008-075 WO 1

[0096] In certain embodiments, the stationary phase is contained within a filter, such as, e.g., EMPHAZE™”, where the filter can be a single filter or can be connected (i.e., “stacked”) in a series, for example, to accommodate the loading density of the target biomaterial contained in the loading solution. For example, to determine whether a given loading density is suitable for a given filter to be used for separation of a biomaterial mixture containing a particular target biomaterial, the skilled artisan can test the efficiency of separation of the biomaterial mixture during an exploratory displacement chromatography run. For example, if the resulting yield of the purified target biomaterial is low but the purity is high, conditions can be adjusted to load more target biomaterial per filter, for example, by stacking fewer filters per process run. Conversely, if the yield is high but the purity is low, conditions can be adjusted to load less target biomaterial per filter, e.g., by stacking more filters per process run. Such optimization process is well within the skill in the art and can be repeated until an optimal loading density is determined in conjunction with the available displacement molecules in the pool (e.g., HMWIs). Other process parameters can be similarly adjusted and optimized.

[0097] Displacement Enhancer: Displacement enhancers suitable for use in the aqueousbased chromatography methods provided herein are any non-naturally occurring (i.e., synthetic), non-ionic, water-soluble polymer, although naturally occurring, water- soluble, non-ionic polymers such as, for example, polymers composed of repeating sugars and plant-based polymers, such as, for example, amylose, dextrans, and glycogen are contemplated for use as well. Suitable polymers are non-reactive (e.g., chemically inert), e.g., under standard chromatographic separation and processing conditions. Representative polymers include, e.g., poly(ethylene glycol) (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and the like, and copolymers or combinations thereof. The polymer can possess a linear or branched geometry; polymers for use in the chromatographic separation method provided generally but not necessarily have an average molecular weight from about 300 to about 100,000 daltons. Higher molecular weight poly(ethylene glycols) typically have a higher viscosity than lower molecular weight poly(ethylene glycols); the enhancement displacer concentration and other operating conditions for the chromatographic separation can therefore be adjusted accordingly, depending upon the components of the biomaterial mixture. The non-naturally occurring, non-ionic water-soluble polymer can possess an average molecular weight falling within the following illustrative ranges: from about 300IGM Ref. No. 008-075 WO 1 daltons to about 40,000 daltons, from about 350 daltons to about 30,000 daltons, from about 350 daltons to about 20,000 daltons, from about 350 daltons to about 10,000 daltons, from about 350 daltons to about 8,000 daltons, from about 350 daltons to about 6,000 daltons, and from about 400 daltons to about 5,000 daltons. Additional representative molecular weight ranges include, e.g., from about 500 daltons to about 10,000 daltons, from about 600 daltons to about 15,000 daltons, from about 600 daltons to about 6000 daltons, and from about 600 daltons to about 5000 daltons. Additional representative molecular weight ranges include, e.g., from about 5,000 daltons to about 100,000 daltons, from about 10,000 daltons to about 80,000 daltons, and from about 15,000 daltons to about 60,000 daltons. Polymers falling within the above molecular weight ranges are commercially available or can be readily supplied by a polymer manufacturer upon request. Illustrative polymer average molecular weights include 400, 500, 1,000, 1,500, 3,000, 4,000, 4,600, 5,000, 6,000, 8,000, 10,000, 12,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, and 80,000 daltons or greater. One illustrative non-naturally occurring, non-ionic, water-soluble polymer for use in the chromatographic methods provided herein is poly(ethylene glycol) (PEG). PEGs comprise ethylene oxide repeat units, ~(CH2CH2O)n~, where “n” indicates the average number of repeat units. For example, a PEG having an average molecular weight from about 300 to about 100,000 daltons corresponds to PEGs comprising the above formula (i.e., a series of monomer repeat units) with “n” values from about 6 to about 2273. Poly(ethylene glycols) for use as a displacement enhancer possess non-reactive terminal functional groups, such as, for example, hydroxy and / or methoxy or any other suitable non-reactive end-capping group. Poly(ethylene glycol) having terminal hydroxy end groups, when in linear form, is also sometimes referred to as “PEG diol”, having a formula, HO(CH2CH2O)nH. Other suitable non-reactive terminal functional groups (also referred to as end-capping groups) include lower alkoxy (i.e., Ci-Ce alkoxy) groups, such as, for example, methoxy, ethoxy, propyloxy, etc. (CH3(CH2)mO~), where m=0, 1, 2, 3, 4, or 5. In some embodiments of the method, the PEG comprises hydroxy and / or methoxy terminal end groups such as PEG diol or methoxy PEG (also referred to as mPEG or poly(ethylene glycol) methyl ether, e.g., HO(CH2CH2O)nCH3) for use as a displacement enhancer in the methods provided herein (where the value of n corresponds to an integer corresponding to one or more of the molecular weight ranges or average molecular weights described above). Branched PEGs (or other non-naturally occurring,IGM Ref. No. 008-075 WO 1 non-ionic, water-soluble polymers, e.g., PEGs having two, three, or four or more arms) are also suitable for use as a displacement enhancer. Commercially available branched polymers generally possess one or more reactive chemical groups, e.g., for conjugation, which can readily be transformed into a non-reactive chemical group(s) using standard organic chemistry techniques. A wide variety of branched poly(ethylene glycols) are available from, for example, NOF America Corporation, JenKem Technology USA, and Creative PEGWorks. It is to be understood that descriptions related to the use of PEG as an exemplary displacement enhancer are also meant to equally apply to and encompass other non-naturally occurring, non-ionic, water-soluble polymers unless noted otherwise.

[0098] Loading Solution / Salts and Buffers: The chromatographic separation is carried out in an aqueous solution (i.e., the loading solution) comprising one or more salts. Salts for use in the method are those comprising ions that are not denaturing. Exemplary salts are non-chaotropic. Any of a number of salts can be used, for example, mineral salts, organic salts, including buffer salts, or a combination thereof. Thus, in some embodiments, the salt is not a buffer salt, such as, for example, sodium or potassium chloride. In some other embodiments, the salt is a buffer salt. In some further embodiments, the aqueous solution comprises a non-buffer salt, such as, for example, sodium chloride, and a buffer salt. Salts suitable for use in aqueous chromatography for separating biomolecules are well-known in the art. Salts that can be comprised in the aqueous loading solution include, for example, sodium chloride, sodium phosphate, sodium acetate, sodium sulfate, sodium citrate, potassium chloride, potassium phosphate, potassium acetate, potassium sulfate, potassium citrate, tris chloride or a combination of one or more of the foregoing. Buffer salts that can be comprised in the loading solution include ionic forms of any of the Good’s buffers with a suitable counterion, such as, for example, salt forms of MES, bis-tris, ADA, ACES, PIPES, MOPSO, bis-6-tris propane, bicine, BES, MOPS, TES, HEPES, DIPSO, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, tricine, glycylglycine, glycinamide, and CABS. Additional buffers / buffer salts for use in the method include, e.g., cholamine chloride hydrochloride, histidine / histidine chloride, imidazole / imidazolium chloride, succinic acid / sodium succinate, glutamic acid / sodium glutamate, glycine / glycine hydrochloride, ammonia / ammonium chloride, boric acid / sodium borate, and Britton-Robinson buffer. In some embodiments of the method, a salt comprised in the loading solution is sodium chloride, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, tris chloride or a combination ofIGM Ref. No. 008-075 WO 1 one or more of the foregoing. In some other embodiments, a salt comprised in the loading solution is a salt form of a buffer or buffer component. In some particular embodiments, the loading solution comprises sodium chloride and sodium phosphate.

[0099] In some embodiments of the method, the aqueous loading solution comprises a buffer. Buffer solutions typically contain a mixture of a weak acid and its conjugate base or vice versa, and as such, depending upon the pH of the loading solution, a salt comprised in the loading solution can also act as a buffer. Illustrative biological buffers suitable for use in a polymer-assisted displacement chromatography method as provided herein include, e.g., those buffers described above, such as phosphate buffers, histidine, imidazole, Good’s buffers, or a combination of one or more of the foregoing. Phosphate buffers suitable for use include, e.g., sodium phosphate and potassium phosphate buffers; Goods buffers include, e.g., MES buffer, bis-tris buffer, ADA buffer, ACES buffer, PIPES buffer, MOPSO buffer, bis-6-tris propane buffer, BES buffer, MOPS buffer, TES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, TAPSO buffer, HEPPSO buffer, POPSO buffer, HEPPS buffer, tricine buffer, Gly-Gly buffer, bicine buffer, HEPBS buffer, TAPS buffer, AMPD buffer, TABS buffer, AMPSO buffer, CHES buffer, CAPSO buffer, AMP buffer, CAPS buffer, and CABS buffer. In some embodiments, a buffer comprised in the loading solution is sodium phosphate. Such buffers are commercially available or can be readily prepared and are well-known in the art. The buffer is typically employed at a concentration sufficient to maintain its buffering capacity during the polymer-assisted displacement chromatography process.

[0100] Typically, the pH of the loading solution ranges from about 4.0 to about 10, depending upon the components of the biomaterial mixture, including, e.g., the target biomaterial, and factors related thereto, such as, for example, susceptibility to deamidation, unfolding, denaturing, aggregation, precipitation, charge, etc., as well as the particular chromatography medium employed. Exemplary pH ranges of the loading solution include, for example, from about 5.0 to about 10, or from about 5.0 to about 9, or from about 5.0 to about 8.6. That is, in some embodiments, the pH of the loading solution is about 5.0, 5.2, 5.5, 5.7, 6.0, 6.2, 6.5, 6.7, 7.0, 7.2, 7.5, 7.7, 8.0, 8.2, 8.5, 8.7, or has a value among or between any two of the foregoing pHs. In some embodiments, the pH of the loading solution is in a range from about 7.0 to about 7.8; in some other embodiments, the pH of the loading solution is about 7.0.Biomaterial Mixture ComponentsIGM Ref. No. 008-075 WO 1

[0101] Target biomaterials: As described above, the polymer-assisted displacement chromatography method is useful for separating components of a biomaterial mixture comprising one or more high molecular weight biomaterial impurities (HMWI) and a target biomaterial having a lower molecular weight than the HMWI. Target biomaterials include, for example, peptides, polypeptides, proteins, polynucleotides, and other macromolecules in non-aggregated form. In some embodiments, the target biomaterial is a protein. In some embodiments, the target material is a monoclonal antibody. In some other embodiments, the target biomaterial is an antibody or an antibody fragment, such as, for example, a Fab fragment, a scFv, a diabody or a triabody. In yet some particular embodiments, the antibody is a multimeric antibody such as, for example, an IgA or an IgM antibody.

[0102] The methods described herein can be used to purify, e.g., polypeptide preparations that include polypeptides, e.g., antibodies, from natural, synthetic, or recombinant sources. Antibody preparations can come from, for example, ascites, bacterial lysates, conditioned cell culture media, cell-free media, milk, plant extracts, plasma, serum, or yeast lysates. Prior to the polymer-assisted displacement chromatography method provided herein, the biomaterial mixture can undergo one or more upstream pre- purification / processing steps, e.g., chromatography, precipitation, or other fractionation step, or combination thereof. See FIG. 2. For example, upstream chromatography steps can employ conventional affinity, e.g., protein A (for IgG antibodies), immobilized metal, lectin, immune-affinity, anion exchange, cation exchange, hydrophobic interaction, size exclusion, and / or mixed-mode chromatography. Precipitation can comprise, for example, salt or PEG precipitation. Fractionation steps can include, e.g., crystallization or membrane filtration. One or more upstream pre-purification steps of a biomaterial mixture are not limited in this regard.

[0103] Examples of polypeptides that can be purified by the methods provided herein include, for example, immunoglobulins, antibodies, enzymes, hormones, fusion proteins, blood factors, growth factors, anti -coagulants, immunoconjugates, interferons, thrombolytics, bone morphogenic proteins, and cytokines.

[0104] Antibodies to be purified by the present methods can be of any derivation (e.g., murine, leporine, simian, human, etc.). In certain embodiments, the antibodies comprised in the biomaterial mixture are human or chimeric human / mouse antibodies. The antibodies can be of any isotype (e.g., IgA, IgG, IgM, etc.), and the disclosure is notIGM Ref. No. 008-075 WO 1 limited in this regard. In some embodiments of the method, the target biomaterial is a multimeric binding molecule. Exemplary multimeric binding molecules include, for example, a multimeric antibody comprising two, four, five, or six bivalent binding units, wherein each binding unit comprises two heavy chains each comprising at least the heavy chain variable region of a binding domain and an IgA heavy chain constant region or multimerizing fragment or variant thereof or an IgM heavy chain constant region or multimerizing fragment or variant thereof. In some particular embodiments of the method, the target biomaterial is a pentameric antibody. In some other embodiments, the target biomaterial is a hexameric antibody. In yet some other embodiments of the method, the antibody is a tetrameric antibody. In some further embodiments, the target biomaterial is an IgA antibody and the chromatography medium is a cation exchange medium. In yet some further embodiments, the target biomaterial is a pentameric or a hexameric IgM antibody and the chromatography medium is an anion exchange medium.

[0105] In certain embodiments, the antibodies to be purified using the polymer-assisted displacement chromatography methods provided herein are IgA antibodies or IgM antibodies. Where the antibodies to be purified are IgM antibodies, such antibodies can contain a modified IgM constant region that includes one or more amino acid substitutions relative to a wild-type IgM constant region as described in U.S. Patent No. 11,401,337. Where the antibodies to be purified are IgA or IgM antibodies, they can, in some embodiments, comprise a J chain. Such antibodies can comprise a modified J chain, that is, a J chain that has been modified by introducing one or more binding moieties into a J chain sequence as described in U.S. Patent No. 10,400,038. IgA or IgM antibodies to be purified can also comprise a J chain that has been modified to comprise one or more ADME (absorption, distribution, metabolism and / or excretion)-modulating moieties into a J chain sequence as described in, for example, U.S. Patent No.10,618,978. In some particular embodiments, the antibody to be purified is a pentameric, human IgM antibody with a modified J chain as described above. For example, illustrative antibodies are described in U.S. Patent Nos. 9,951,134 and 10,618,978. Additionally, IgM antibodies to be purified can comprise a variant J chain or functional fragment thereof, where the variant J chain (or fragment thereof) includes one or more single amino acid substitutions, deletions, or insertions relative to a reference J chain identical to the variant J chain except for the one or more single amino acid substitutions, deletions, or insertions, where the variant J chain can, for example, increase the serumIGM Ref. No. 008-075 WO 1 half-life of the provided IgM antibody or IgM-like antibody. Such illustrative antibodies and variant J chains are described in U.S. Patent No. 10,899,835. Illustrative antibodies and variant J chains can comprise, for example, an alanine (A) or arginine (R) substitution at a position corresponding to amino acid Y102 of the wild-type human J chain, an alanine (A) substitution at a position corresponding to amino acid N49 of the wild-type human J chain, or an alanine (A) or arginine (R) substitution at a position corresponding to amino acid Y102 of the wild-type human J chain.

[0106] In some embodiments, the antibody comprised in a biomaterial mixture to be purified is an anti-CD20 x anti-CD3s bispecific, pentameric IgM, such as, e.g., imvotamab (also referred to as IGM-2323). Imvotamab is a bispecific antibody based on an engineered pentameric IgM framework, with a recombinant J-chain that is fused to both an anti-CD3 scFv and also to human serum albumin. Illustrative dimeric, pentameric and hexameric binding molecules specific for CD20 and CD30 are described, for example, in U.S. Patent Nos. 10,787,520 and 10,618,978.

[0107] Further illustrative antibodies are anti-CD38 antibodies such as, for example, the multimeric antibodies (comprising 2, 4, 5, or 6 bivalent binding units) described in WO 2023 / 150677. In some particular embodiments, the antibody is an anti-CD38 x anti- CD3s antibody. In certain embodiments, the antibody is a bispecific, pentameric IgM, e.g., IGM B-2 as described in WO 2023 / 150677.

[0108] Additional illustrative antibodies for chromatographic purification according to the methods provided herein are antibodies directed against death receptor 5 (DR5), also known as TRAIL receptor 2. In some embodiments, the anti-DR5 antibody to be purified is a multimeric antibody, e.g., a dimeric, pentameric or hexameric binding molecule with 2, 5, or 6 bivalent binding units, that specifically and agonistically binds to DR5, e.g., as described in U.S. Patent Nos. 9,938,347 and 10,689,449. In certain embodiments, the antibody is a pentameric anti-DR5 IgM antibody such as, e.g., anti-DR5 Mab No. 1, No. 2, No. 3 and No. 4 as described in U.S. Patent No. 9,938,347. In some particular embodiments, the pentameric anti-DR5 antibody is aplitabart.

[0109] In yet some further embodiments, the antibody is a multimeric SARS-CoV2 binding molecule. In some embodiments, the multimeric SARS-CoV2 binding molecule is a binding molecule as described in U.S. Patent Publication No. US 2022 / 0267415. In some particular embodiments, the antibody is a pentameric IgM directed against SARS-IGM Ref. No. 008-075 WO 1 CoV2 spike protein receptor binding domain. Illustrative anti-SARS-CoV-2 pentameric IgM antibodies are described in U.S. Patent Publication No. US 2022 / 0267415.

[0110] Additional exemplary antibodies for purification according to the methods provided herein are antibodies that specifically bind to CD 123, such as those antibodies described in International Patent Application Publication No. WO 2022 / 178047. In some embodiments, an antibody to be purified is a bispecific, pentameric IgM antibody with ten binding domains that bind to CD 123 and a single anti-CD3s binding domain on the J chain, e.g., 1132716-1-1 IgM as described in WO 2022 / 178047.[OHl] Additional illustrative antibodies for chromatographic purification according to the methods provided herein are immunostimulatory multimeric binding molecules such as those described in WO 2021 / 030688. In some embodiments, the multimeric binding molecule is a dimeric or pentameric IgM comprising binding domains that specifically bind to and antagonize PD-L1, where the binding molecule further comprises a human variant J chain comprising an immunostimulatory agent comprising an interleukin- 15 protein and an IL-15 receptor-a fragment comprising the sushi domain. In some particular embodiments, the multimeric binding molecule is, for example, PDL1-IL-15 IgMl as described in WO 2021 / 030688.

[0112] Further exemplary antibodies for chromatographic purification according to the methods provided herein are T cell costimulatory multimeric binding molecules such as those described in WO 2024 / 148336.

[0113] Additional exemplary antibodies for polymer-assisted displacement chromatographic purification as provided herein include IgG antibodies having binding domains as described for the illustrative multimeric antibodies described above.

[0114] As described previously, the chromatographic methods provided herein are useful for separating high molecular weight impurities from a biomaterial mixture comprising a target biomaterial, such as, for example, a peptide, protein, polynucleotide, or other macromolecules, and the disclosure is not limited in this regard.

[0115] High Molecular Weight Impurity (HMWI): The polymer-assisted displacement chromatography methods provided herein are effective to largely remove HMWIs from the aqueous loading solution. HMWIs can form during protein production; formation of such HMWIs, if not substantially removed, can, for example, compromise the effectiveness of a biopharmaceutical product, increase the risk of an immune response upon administration, and pose a safety concern. Thus, innovative methods and strategiesIGM Ref. No. 008-075 WO 1 such as the methods provided herein are needed to mitigate the potential adverse consequences of having undesirable amounts of HMWIs present in protein-based therapeutic products, e.g., by substantially removing such materials during manufacturing.

[0116] HMWIs include, for example, high molecular weight species such as aggregated proteins, host-cell proteins, inclusion bodies, endotoxins, viruses, liposomes, nucleic acids, plasmids, and the like, and combinations thereof, where, in accordance with the displacement methods provided herein, the HMWI has a higher molecular weight than the target biomaterial (or, as stated otherwise, the target biomaterial has a lower molecular weight than the HMWI). Protein aggregates form when disordered or misfolded proteins aggregate. Proteins formed from mammalian cell culture can contain significant amounts of dimers, trimers, and other higher order aggregates. Host cell proteins (HCPs) are process-related impurities that can arise during protein production from mammalian cell lines; the presence of HCPs or other high-molecular weight impurities can lead to fragmentation of the target biomaterial, immunogenic responses and changes in drug product formulations. Viral contaminants pose a safety risk; four of the five viruses found to contaminate human and primate cell lines, herpes virus, human adenovirus type 1, parainfluenza virus type 3 and reovirus type 3, are known to be pathogenic in humans, whereas one of the viruses, Cache Valley virus, has been reported to cause disease in humans (Barone, P.W., et al., Nature Biotechnology, 38, May 2020, p. 563-572). As shown in the supporting examples, the polymer-assisted displacement chromatography methods provided herein are effective to provide highly purified recovered target biomaterial, and demonstrate highly efficient and effective removal of different types of HMWIs such as, for example, aggregated protein (from a variety of antibody preparations, see, e.g., Examples 1, 2, 3, 4, 7, 8, 10), host-cell protein (Example 5), and virus (Examples 6, 9).

[0117] Polymer- Assisted Displacement Chromatography Method: The method provided herein comprises applying an aqueous loading solution comprising a biomaterial mixture comprising components as described above (i.e., a target biomaterial and HMWI), a salt, and a displacement enhancer (i.e., a non-naturally occurring, non-ionic water-soluble polymer) to a chromatography medium, and continuing to apply the aqueous loading solution to the chromatography medium to effect displacement of the target biomaterial from the chromatography medium. Incorporation of the displacement enhancer into theIGM Ref. No. 008-075 WO 1 loading is effective to promote displacement of the target biomaterial from the chromatography medium with selective retainment of high molecular weight impurities on the chromatography medium over the target biomaterial, to thereby remove, to a large extent, such high molecular weight impurities from the biomaterial mixture. That is, in the presence of the displacement enhancer, intact, non-aggregated target biomolecules are out-competed and selectively displaced by the HMWIs which are then largely retained on the chromatography medium to provide an eluate comprising the target biomaterial at a higher purity relative to that of the loading solution. See, for example, the accompanying examples.

[0118] The aqueous loading solution, of a given volume, is continuously applied to the chromatography medium - in the absence of an elution gradient - to allow the chromatography medium to become saturated with the target biomaterial, followed by displacement of the target biomaterial from the chromatography medium, wherein the displacement is enhanced due to the presence of the non-naturally occurring, water- soluble, non-ionic polymer, e.g., poly(ethylene glycol). Eluate samples comprising the target biomaterial (i.e., at a higher purity relative to that of the loading solution, e.g., reduced in HMWI content) are collected following elution through the chromatography medium.

[0119] Illustrative aqueous loading solutions comprise, for example, from about 0.1% (w / v) to about 10% (w / v) of a non-naturally occurring, non-ionic, water-soluble polymer (i.e., the displacement enhancer), e.g., poly(ethylene glycol). The loading solution can contain, for example, from about 0.1% to about 1% displacement enhancer, from about 0.1% to about 2% displacement enhancer, from about 0.1% to about 3% displacement enhancer, from about 0.2% to about 2% displacement enhancer, from about 0.3% to about 3% displacement enhancer, from about 0.5% to about 5% displacement enhancer, from about 0.5% to about 10% displacement enhancer, from about 0.5% to about 3% displacement enhancer, from about 0.5% to about 2% displacement enhancer, from about 1% to about 3% displacement enhancer, from about 1% to about 5% displacement enhancer, from about 1% to about 7% displacement enhancer, or from about 1% to about 10% displacement enhancer (w / v). In some embodiments, the loading solution contains about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% (w / v) displacement enhancer, inclusive of any and all ranges between any two of the foregoing.IGM Ref. No. 008-075 WO 1

[0120] Exemplary biomaterial mixtures to be separated contain, for example, from about 20 percent to about 1 percent or less HMWI (e.g., aggregated protein, a host-cell protein, virus, nucleic acid, plasmid, liposomes, etc.) of total biomaterial in the loading solution, from about 18 percent to about 3 percent HMWI of total biomaterial in the loading solution, from about 15 percent to about 7 percent HMWI of total biomaterial in the loading solution, from about 14 percent to about 2 percent HMWI of total biomaterial in the loading solution, or from about 12 percent to about 3 percent HMWI of total biomaterial in the loading solution (w / v), inclusive of all ranges falling between members of each of the foregoing ranges. In some embodiments, the loading solution comprises HMWI and is substantially free of host cell protein, such that host cell protein is below the assay limit of detection. For loading solutions comprising host cell protein, in some embodiments, the host cell protein content of the loading solution does not exceed 50,000 parts per million (ppm), does not exceed 10,000 parts per million, or does not exceed 4,000 parts per million host cell protein, where “does not exceed” includes a lower limit of host cell protein of about 10 ppm. See, for example, Example 5, which demonstrates the effectiveness of the instant method (see Table 5, “Emphaze” row) at removing host cell proteins from an exemplary biomaterial mixture in comparison to other chromatographic purification steps. Considering each of the three illustrative purification runs, the polymer-assisted displacement method disclosed herein was effective to achieve from approximately a 90-fold reduction in host cell protein to about a 730-fold reduction in host cell protein, which illustrates a degree of removal of host cell protein that was significantly higher than achieved in any of the other chromatographic purification steps explored.

[0121] In some embodiments, the biomaterial mixture comprises a virus as a high molecular weight impurity, and the polymer-assisted displacement method disclosed herein results in a large degree of viral clearance, for example, from about 2 logs to about 7 logs of virus clearance from the biomaterial mixture, or from about 3 logs to about 7 logs of virus clearance from the biomaterial mixture, or from about 4 logs to about 7 logs of virus clearance from the biomaterial mixture, or from about 3 logs to about 6 logs of virus clearance from the biomaterial mixture, or from about 3 logs to about 5 logs of virus clearance from the biomaterial mixture. See, for example, Example 6, illustrating successful removal of an exemplary virus from a biomaterial mixture comprising an IgM antibody, wherein virus capture onto the chromatography mediumIGM Ref. No. 008-075 WO 1 was confirmed following purification via polymer-assisted displacement chromatography.

[0122] In terms of loading density, generally, the loading solution is applied to the chromatography medium at a loading density of from about 15 mg / mL to about 350 to 400 mg / mL target biomolecule, from about 20 mg / mL to about 300 mg / mL target biomolecule, from about 25 mg / mL to about 200 mg / mL target biomolecule, from about 25 mg / mL to about 100 mg / mL target biomolecule, from about 20 mg / mL to 80 mg / mL target biomolecule, from about 20 mg / mL to 50 mg / mL target biomolecule, from about 40 mg / mL to 100 mg / mL target biomaterial, from about 50 to about 95 mg / mL target biomaterial, or from, e.g., about 60 mg / mL to about 90 mg / mL of target biomaterial, depending, for example, upon the features of the target biomolecule and scale of the process. An aqueous loading solution, as used herein, comprises the biomaterial mixture that is loaded onto the chromatography medium for the polymer-assisted chromatography process provided herein.

[0123] Alternatively, loading density can refer to the biomaterial mixture that is loaded onto the chromatography medium. Exemplary loading densities also include the following. For example, the loading solution can be applied to the chromatography medium at a loading density of from about 15 mg / mL to about 350 to 400 mg / mL biomaterial mixture, from about 20 mg / mL to about 300 mg / mL biomaterial mixture, from about 25 mg / mL to about 200 mg / mL biomaterial mixture, from about 25 mg / mL to about 100 mg / mL biomaterial mixture, from about 20 mg / mL to 80 mg / mL biomaterial mixture, from about 20 mg / mL to 50 mg / mL biomaterial mixture, from about 40 mg / mL to 100 mg / mL biomaterial mixture, from about 50 to about 95 mg / mL biomaterial mixture, or from, e.g., about 60 mg / mL to about 90 mg / mL biomaterial mixture.

[0124] The polymer-assisted displacement chromatography is typically carried out at a pH of the loading solution from about 4.0 to about 10, depending upon the structure and properties of the target biomaterial. Illustrative pH ranges of the loading solution are from about 4.5 to about 10, from about 5.0 to about 9.0, from about 5.5 to about 8.6, from about 5.0 to about 8.6, from about 6.0 to about 7.8, with additional illustrative pHs falling within one of the foregoing ranges. In some particular embodiments, the pH of the loading solution is from about 6.5 to about 7.8.IGM Ref. No. 008-075 WO 1

[0125] As described above, the loading solution typically comprises a salt, e.g., at a concentration of at least about 50 mM (where exemplary salts are described elsewhere herein), or, in some embodiments, at a concentration of at least about 100 mM. In some embodiments, the concentration of salt in the loading solution is in a range of from about 75 mM to about 500 mM, with further illustrative salt concentrations ranging from about 80 mM to about 450 mM, from about 100 mM to about 400 mM, and from about 100 mM to about 300 mM. In some embodiments, the salt is a non-buffer salt, such as, for example, sodium chloride. In other embodiments, the salt is a non-buffer salt such as, for example, potassium chloride. In some embodiments, the aqueous loading solution comprises at least about or about 125 mM salt, at least about or about 150 mM salt, at least about or about 175 mM salt, at least about or about 200 mM salt, at least about or about 225 mM salt, at least about or about 250 mM salt, at least about or about 275 mM salt, at least about or about 300 mM salt, at least about or about 325 mM salt, or even at least about or about 350 mM salt.

[0126] In some other embodiments, the loading solution comprises a buffer salt (i.e., a buffer). In some embodiments, the loading solution comprises both a non-buffer salt and a buffer salt. In some embodiments, the concentration of buffer salt in the loading solution ranges from about 2 mM to about 100 mM, or even greater. For instance, exemplary concentrations of buffer salt in the loading solution can range from, for example, about 5 mM to about 90 mM, from about 10 mM to about 80 mM, from about 15 mM to about 70 mM, from about 20 mM to about 60 mM, from about 2 mM to about 25 mM, from about 5 mM to about 15 mM, from about 7 mM to about 12 mM, from about 8 mM to about 11 mM. In some particular embodiments, the concentration of buffer salt in the loading solution ranges from about 8 mM to about 11 mM, e.g., about 10 mM. An equilibration buffer (also referred to as an equilibrium aqueous solution) can be used, for example, to condition the chromatography medium prior to loading of the aqueous loading solution, e.g., by flushing the chromatography medium with the equilibration buffer; a chase buffer (also referred to as an aqueous chase solution) can be used after loading the aqueous loading solution to “chase” out the displaced target biomaterial from the chromatography medium. Typically, during the process, e.g., including equilibration of the chromatography medium by application of an equilibrium buffer, and following the continuous application of the loading solution to the chromatography medium, applying a chase buffer to the chromatography medium toIGM Ref. No. 008-075 WO 1 chase out displaced biomaterial from the chromatography medium, the mobile phase remains more or less constant (buffer salt, pH, polymer, and non-buffer salt remain constant in the mobile phase). For example, in some embodiments, the aqueous loading solution, the equilibration buffer and the chase buffer are the same, with the exception of the biomaterial mixture, which is absent from the equilibrium and chase buffers. Turning to the examples, e.g., an illustrative mobile phase comprises 165 mM NaCl, 3% PEG 4600 w / v, 10 mM NaPi, at pH 7.00, which can be used in an equilibration step, as a loading solution (further comprising the biomaterial mixture to be separated), and applied to the chromatography medium as the chase buffer. The mobile phase can change, though, for example, during a sanitation and / or neutralization preparation of the chromatography medium prior to carrying out the polymer-assisted displacement chromatography method, or following polymer-assisted displacement chromatography, for example, during a cleaning-in-place (CIP) step of the chromatography medium.

[0127] In some embodiments, the loading solution comprises sodium chloride, sodium phosphate and PEG as the displacement enhancer. Additional embodiments of aqueous loading solution components include, e.g., 150-200 mM NaCl. In certain embodiments, the aqueous loading solution further comprises between about 9 mM and about 11 mM of sodium phosphate (e.g., composed of Na2HPO4 and Na^PCU).

[0128] The aqueous loading solution comprising the biomaterial mixture and the displacement enhancer is continuously applied to the chromatography medium, e.g., at a loading density of the chromatography medium that is suitable to achieve the desired quality attributes of the product (recovered target biomaterial). The aqueous loading solution is typically loaded onto the chromatography medium at a loading density that exceeds the binding capacity of the chromatography medium. Illustrative loading densities can range from about, e.g., 10 mg / mL to about 2500 mg / mL, or from about 25 mg / mL to about 2500 mg / mL of total biomaterial, depending upon the chromatography medium, the biomaterial mixture, etc., where the foregoing ranges are intended to be illustrative and in no way limiting. Additional illustrative loading densities include, e.g., from about 50 mg / mL to about 2000 mg / mL total biomaterial, from about 75 mg / mL to about 1500 mg / mL total biomaterial, from about 100 mg / mL to about 1000 mg / mL total biomaterial, from about 100 mg / mL to about 750 mg / mL total biomaterial, and from about 100 mg / mL to about 500 mg / mL total biomaterial. In some embodiments, the biomaterial mixture is loaded onto the chromatography medium at a loading density ofIGM Ref. No. 008-075 WO 1 any of about 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 700 mg / mL, 800 mg / mL, 900 mg / mL, 1000 mg / mL, 1100 mg / mL, 1200 mg / mL, 1300 mg / mL, 1400 mg / mL, 1500 mg / mL, 1600 mg / mL, 1700 mg / mL, 1800 mg / mL, 1900 mg / mL, and 2000 mg / mL or greater, or within a range falling between any two of the foregoing.

[0129] The binding capacity of a chromatography medium for a particular biomaterial (e.g., target molecule and / or high molecular weight impurity) can be determined by evaluating binding, in an iterative fashion, using an excess of a biomaterial. In this way, loading capacity can be optimized for a particular biomaterial mixture, chromatography medium, and loading solution.

[0130] In some embodiments, the chromatography medium is pre-conditioned prior to applying the loading solution. Pre-conditioning steps can include, for example, regeneration and sanitizing of the chromatography medium with a regeneration solution such as, e.g., a sodium hydroxide solution, once compatibility with the chromatography medium and system has been confirmed. Sodium hydroxide can be used to inactivate viruses, bacteria, yeasts, fungi and endotoxins, to dissolve proteins, and to remove nucleic acids. In some embodiments, a salt such as sodium chloride, is added to the sodium hydroxide solution. Following regeneration, the chromatography medium can then be flushed with a suitable neutralization buffer such as, for example, a high concentration phosphate buffer, followed by conditioning with, e.g., an equilibration buffer. In some embodiments, the equilibrium buffer is the same as the loading solution absent the biomaterial mixture.

[0131] In some embodiments, the conductivity of the loading solution is measured prior to loading onto the chromatography medium, e.g., with a conductivity meter. Conductivity refers to the ability of an aqueous solution to conduct an electric current between two electrodes. Since current flows by ion transport in solution, generally, the higher the ionic concentration, the greater the conductivity of the solution. Thus, the conductivity of the aqueous loading solution can be adjusted by changing the concentration of ions (e.g., salt or buffer) therein. Conductivity is measured in milliSiemens per centimeter (mS / cm). In some embodiments, the conductivity of the aqueous loading solution is greater than about any of 5.0 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0IGM Ref. No. 008-075 WO 1 mS / cm, 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 19 mS / cm, and 20 mS / cm. In some embodiments, the conductivity of the aqueous loading solution is between about 10 mS / cm and about 20 mS / cm. In some further embodiments, the conductivity of the aqueous loading solution is between about 20 mS / cm and about 35 mS / cm. In some other embodiments, the conductivity of the aqueous loading solution is between about 12 mS / cm and about 19 mS / cm. In yet some further embodiments, the conductivity of the aqueous loading solution is between about 17-19 mS / cm. If the conductivity is greater than a desired value, the aqueous loading solution can be suitably diluted to arrive at a desired conductivity. If the conductivity is less than a desired value, the ionic strength of the aqueous loading solution can be increased by addition of salt and / or buffer until a desired conductivity is reached. The conductivity of the aqueous loading solution can be adjusted to favor binding of both the target biomaterial and the high molecular weight impurities to the chromatography medium in the presence of the displacement enhancer, e.g., PEG.

[0132] Once the conductivity of the aqueous loading sample is in a desired range, the aqueous loading solution is continuously loaded onto the chromatography medium. The flow rate can be adjusted to achieve a desired residence time, e.g., based upon chromatography medium bed height or chromatography medium volume and flow velocity. The residence time, also referred to as retention time, is a measure of time taken for a solute to pass through a chromatography medium, and can vary widely depending upon the chromatography medium, column or filter size, and acceptable loading, among other things. Suitable residence times can be determined by the skilled artisan. No elution gradient is required; in the presence of the displacement enhancer, following saturation of binding sites on the chromatography medium with the target molecule, nonaggregated target biomolecules are out-competed and selectively displaced from the chromatography medium by the HMWIs, which are then largely retained on the chromatography medium to provide an eluate comprising the target molecule at a higher purity relative to that of the loading solution. More particularly, as the aqueous loading solution is continuously applied to the chromatography medium, bound target molecules are displaced from the chromatography medium as the loading solution continues to pass through the chromatography medium.IGM Ref. No. 008-075 WO 1

[0133] The aqueous solution containing displaced target molecule is collected, typically in fractions. Fraction collection is typically commenced upon detection of target molecule. Once all of the loading solution has been applied to the chromatography medium, an aqueous wash (chase) buffer can then be applied to the stationary phase to push out remaining target molecule that remains (e.g., is loosely bound) on the chromatography medium. The wash (chase) buffer will typically, but not necessarily, be of about the same composition (with the exception of the biomaterial mixture components), and / or pH, and and / or conductivity as the loading solution. In some embodiments, the wash (chase) buffer is of about the same composition (with the exception of the biomaterial mixture components), pH, and conductivity as the loading solution. Elution conditions for the target molecule from the chromatography medium can be optimized, e.g., to minimize elution of HMWI, or to minimize tailing of the elution profile, by varying operating parameters such as, e.g., salt concentration, amount of displacement enhancer, buffer components, biomaterial mixture concentration, pH, conductivity, flow rate and the like, and making suitable adjustments. Once elution of the target molecule has commenced, the earliest eluting fractions will typically contain substantially pure preparations of the target molecule, i.e., such fractions will typically comprise higher purity target molecule, with low or minimal amounts of HMWI, when compared to the aqueous loading solution. Eventually, the chromatography medium will become saturated with the HMWI (e.g., aggregated protein, virus, etc.), at which point the collected fractions will typically begin to show an increase in the amount of HMWI. At this point, the operator can cease sample collection and flow of solvent (e.g., wash buffer) through the chromatography medium.

[0134] In some embodiments, the method is effective to remove at least 65 percent of the HMWI contained in the biomaterial mixture comprised in the aqueous loading solution. In some further embodiments, the method is effective to remove about 65 percent or more, about 70 percent or more, about 75 percent or more, about 80 percent or more, or about 90 percent or more HMWI from the biomaterial mixture, e.g., comprised in the aqueous loading solution, wherein an upper limit of removal is essentially complete removal of HMWI (i.e., to a level that is undetectable using standard analytical methods).

[0135] In some embodiments of the methods disclosed herein, the method further comprises recovering the target biomaterial. In some embodiments, the recovered targetIGM Ref. No. 008-075 WO 1 biomaterial contains less than about 5 percent, less than about 4 percent, less than about 3 percent, or less than about 2 percent HMWI. In yet some further embodiments, the recovered target biomaterial contains no more than about 0.25 percent HMWI. Typically, the amount of HMWI in the recovered target material is determined based upon the area(s) under the curve in a SEC chromatogram of a given sample; that is, by comparing the total peak(s) area attributed to HMWI to the area of the main peak (i.e., target biomaterial) following exclusion of low molecular weight impurities. Low molecular weight impurities are not included in the calculation due to their interference with determining overall HMWI removal efficiency. Other analytical methods for calculating the percentage of HMWI in a recovered target material sample can be used as well. In some other embodiments where the HMWI is a virus, the method results in from about 3 to about 7 logs of virus clearance (e,g., at least about 3 logs of virus clearance when compared to the biomaterial mixture prior to polymer-assisted displacement chromatography, or at least about 4 logs of viral clearance, or at least about 5 logs of viral clearance, or at least about 5.5 logs of viral clearance, or at least about 6 logs of viral clearance, or up to about 7 logs of viral clearance when compared to the biomaterial mixture prior to polymer-assisted displacement chromatography, including ranges of viral clearance between any two of the foregoing log values). The recovered target biomaterial (also referred to as the purified target biomaterial) can, if desired, be concentrated and buffer exchanged following recovery, such as, for example, by ultrafiltration and diafiltration.

[0136] The polymer-assisted displacement chromatography method provided herein can be optimized for the target molecule of interest. For example, process parameters can be optimized for particular molecules (e.g., proteins) to be purified, e.g., to ensure solubility of the target in the presence of the displacement enhancer, and can be assisted by use of in-silico analysis of electrical charge, hydrophobicity, isoelectric point, and modeling of protein regions, etc.

[0137] In some embodiments, the biomaterial mixture can undergo one or more pre- or post-polymer-assisted displacement chromatography purification / processing steps. In some embodiments, the one or more pre-or post-purification steps are chromatography steps. Exemplary pre-purification steps include, e.g., flow-through chromatographic purification steps in which the target biomaterial flows with the eluent through the chromatographic medium while impurities are strongly bound thereto.IGM Ref. No. 008-075 WO 1

[0138] For example, prior to or following the polymer-assisted displacement chromatography method provided herein, the biomaterial mixture can undergo one or more upstream or downstream purification / processing steps, e.g., chromatography, precipitation, or other fractionation step, or combination thereof. See, for example, FIG. 2. Such chromatography steps can employ, for example, conventional affinity chromatography, e.g., protein A chromatography, (for IgG antibodies), protein G chromatography, immobilized metal, lectin, immune-affinity, anion exchange, cation exchange, hydroxyapatite chromatography, hydrophobic interaction, size exclusion, and / or mixed-mode chromatography. Precipitation can comprise, for example, salt or PEG precipitation. Fractionation steps can include, e.g., crystallization or membrane filtration. One or more upstream or downstream purification steps are not limited in this regard.

[0139] In some embodiments, a capture or polishing step is carried out prior to the polymer-assisted displacement chromatography to provide a reduction in process and / or product-related impurities in the load material. In a biomanufacturing process, a capture step typically separates the target molecule from cell debris and other substances in harvested cell culture media. In contrast, a polishing step is a processing step in which impurities or contaminants are removed from a target molecule-containing feed. One or more polishing steps can occur either prior to or after the polymer-assisted displacement chromatography method disclosed herein. Exemplary pre-purification steps can comprise, for example, anion exchange chromatography, cation exchange chromatography, affinity chromatography, or mixed mode chromatography.

[0140] As described above, the polymer-assisted displacement chromatography method is effective to remove both product (e.g., aggregated protein) and process (e.g., host cell protein)-related impurities, including clearance of viruses, to thereby provide highly pure biomolecules, as illustrated in the following illustrative examples. As provided in the following examples, the method can provide high levels of aggregate removal, in some instances, with efficiencies over 90%, can achieve a several hundred-fold reduction in HCP, and highly effective clearance of virus.

[0141] All of the references cited herein are incorporated by reference in their entireties. To the extent that patents or patent applications incorporated by reference herein include definitions that may be considered to contradict the definitions herein, the definitions provided herein are intended to supersede such definitions.IGM Ref. No. 008-075 WO 1

[0142] Many modifications and variations of the methods described herein can be made without departing from the scope of the present disclosure, as will be understood to those skilled in the art. The following examples are offered by way of illustration and not by way of limitation.EXAMPLESMaterials and Methods

[0143] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Hames and Higgins, eds. (1984) Transcription And Translation (IRL Press); Freshney (2016) Culture Of Animal Cells, 7th Edition (Wiley-Blackwell); Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2d Edition (Wiley -Interscience); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); S.C. Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, Vols. 151-155 (Academic Press, Inc., N.Y.); Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Weir and Blackwell, eds.; and in Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0144] General principles of antibody engineering are set forth, e.g., in Strohl, W.R., and L.M. Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are set forth, e.g., in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press).IGM Ref. No. 008-075 WO 1

[0145] General principles of biopharmaceutical production are described, e.g., in Behme, Stefan, Manufacturing of Pharmaceutical Proteins: From Technology to Economy, 34d Ed., 2021 (Wiley-Blackwell).

[0146] Exemplary antibody preparations as described in the following examples were prepared using conventional biotechnology techniques.EXAMPLE 1POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY BISPECIFIC IgM ANTIBODY, IMVOTAMAB

[0147] Chromatographic purification (e.g., using displacement chromatography) of an exemplary protein mixture comprising imvotamab (WHO Drug Information, Vol. 35, No. 4, 2021, List 126, estimated average molecular weight, 976,862 daltons) and imvotamab aggregates (HMWI) was carried out using an AKTA PURE™ chromatography system including a column (0.5 cm diameter x 5 cm bed height, 1 mL bed volume) packed with POROS® HQ 50 Anion Exchange (AEX) resin. Imvotamab is a bispecific IgM antibody targeting CD20 and CD3 proteins. Solvent conditions for loading the imvotamab protein preparation onto the solid phase were selected to favor initial binding of imvotamab and its aggregates to the solid phase.

[0148] The column was first flushed with 3 column volumes of a solution of 1 N NaOH, followed by flushing the column with 500 mM sodium phosphate buffer (NaPi) until the pH stabilized at pH 7.0. Three column volumes of an equilibration buffer containing 10 mM NaPi, 210 mM NaCl, 1.92% PEG (poly(ethylene glycol) - 4600 (Sigma Aldrich) w / v), pH 7.0 were then passed through the column to purge the NaPi and the A280 signal on the ultraviolet (UV) lamp was then zeroed. A 151 mg total protein preparation containing imvotamab and imvotamab aggregates in a NaCl / NaPi buffer was diluted by addition of a PEG displacement enhancer solution (10 mM NaPi, 24% w / v PEG 4600, pH 7.4 -7.7) to increase the concentration of PEG in the preparation to 1.92% (w / v). An aqueous solution of 1 M NaCl / 10 mM NaPi and 10 mM NaPi was added as needed to the protein preparation to maintain the NaCl molarity at approximately 210 mM and the NaPi concentration at approximately 10 mM to achieve a desired conductivity. The conductivity of the protein preparation was measured on a Metler-Toledo conductivity meter to ensure it matched the conductivity of the running buffer. The flow rate wasIGM Ref. No. 008-075 WO 1 adjusted to achieve a 5-minute column residence time and elution of the protein preparation through the column was commenced.

[0149] Sample collection proceeded at fixed fractionation volumes of 4 mL fractions on average. The HMWI content of the displaced pool (i.e., the solution samples collected following passage through the column) and of the material stripped from the column were assayed by SEC-HPLC.

[0150] The results are shown in FIG. 3 (FIG. 3A, B, C, and D) when considered in conjunction with Table 1 below. FIG. 3A is a processing chromatogram illustrating the overall chromatography run, where the X-axis represents both collected fractions and volume in milliliters. FIGs. 3B, 3C, and 3D are size exclusion chromatograms, where the X axis is time, and the peak retention times correspond to the molecular weight of protein species eluting from the column; the peak areas correspond to the relative percentages of the protein species. FIG. 3B shows a peak corresponding to unaggregated (target) antibody present in fraction 1 A3, collected during chromatographic medium saturation during which protein in the sample saturates sites on the chromatography medium. This fraction, 1 A3, contained only 0.11% of the total antibody yield and only about 0.0022% of aggregates. As elution continued and more fractions were collected following saturation of the chromatographic medium and displacement of nonaggregated (i.e., target) protein, as can be seen in FIG. 3C, the samples became enriched in total antibody yield until the chase portion of the method (Fraction 1C3) exited the column. Fractions 1A4-1C3 (collected and combined) contained 92% of the total antibody yield, but only 45.32% of the aggregate, demonstrating that the chromatography method as provided herein results in the recovery of samples that are considerably purer with respect to the target protein, i.e., non-aggregated antibody, in the displaced pool relative to the loading solution - with a high percentage of aggregates remaining bound to the chromatography medium. After the chromatography run was completed, the column was stripped to analyze the material retained on the column using 1 M NaCl, 10 mM NaPi, pH 7.00). FIG. 3D shows the relative content of the “strip” fraction (the material that remained on the column). It was determined that the strip fraction contained only 1.5% of the total monomeric antibody yield, but contained 42.02% of the aggregated protein, demonstrating that the aggregate remained preferentially bound to the chromatography medium during the course of elution of the protein sample preparation through the resin.IGM Ref. No. 008-075 WO 1

[0151] Fraction-by-fraction results of the exemplary chromatography run described above are summarized in Table 1.* represents percentage reduction in HMWI relative to preceding sample. For example, fraction 1A3 contains 97.6% less aggregate by percentage than the starting sample containing 13.2% aggregate [100% - (0.3% HMWI / 13.2% HMWI) = HMWI reduction of 97.6%]. Typically, LMW’s are excluded from the integration in order to analyze aggregate removal efficiency by utilizing the HMWI to Target protein ratio.

[0152] The chromatography run conditions, solvents, stationary phase, scale, particular displacement enhancer molecule, displacement enhancer molecule molecular weight, protein or other molecule to be purified, high molecular weight biomaterial impurities, and the like, as provided in these examples, are meant to be exemplary. The purification process as provided herein can be carried out on any of a number of target biomaterials and under a variety of conditions as detailed elsewhere herein to remove undesirable high molecular weight impurities and provide a purified target biomaterial product in a highly efficient and reproducible manner.EXAMPLE 2POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY BISPECIFIC IGM ANTIBODY, IMVOTAMAB, AT A MULTI-GRAM SCALE

[0153] Two sample preparations both containing imvotamab and imvotamab aggregates were prepared for chromatographic purification using PEG as a displacement enhancer in each of the protein samples. Sample 1 contained 150 mg total protein, while Sample 2IGM Ref. No. 008-075 WO 1 contained 13.7 g total protein (a scale-up of over 100-fold). The samples were prepared by addition of a NaPi / NaCl solution containing PEG (10 mM NaPi, 165 mM NaCl, 24% w / v PEG 4600, pH 7.0) to both total protein samples, to adjust the concentration of PEG 4600 in each sample to 3% (w / v) while keeping the molarity of NaCl and NaPi and the pH constant. The two preparations were each purified using an AKTA PURE™ chromatography system loaded with a 3M EMPHAZE™ AEX filter. Parameters related to purification of the two preparations are provided in Table 2 below.

[0154] By way of explanation, in considering the load density for Sample 1, the sample was applied to the AEX filter at 36 mg total / mL bed volume (aggregates and nonaggregates) and 30 mg target / mL bed volume (i.e., monomer), where an aim of the process is to provide a high non-aggregated protein (target) recovery yield and removal of aggregates or other HMWIs.*represents reduction in aggregate mass relative to starting sample. For example, if starting sample contains 10 mg of HMWI, and end sample contains 1 mg of HMWI, HMWI removal efficiency is 90% [100% - (1 mg / 10 mg) = 90% aggregate removal]. Typically, LMW’s are excluded from the integration in order to analyze aggregate removal efficiency by utilizing the HMWI to Target protein ratio.

[0155] In the following size exclusion chromatograms, FIG. 4A shows an analysis ofSample 1 before purification, where the aggregate component amounted to 11.04% of the total protein content. FIG. 4B provides the results of the purification following elution of the protein preparation through the filter, where, for recovered protein, aggregate amounted to only 1.38% of the total protein content in Sample 1. Following stripping of the filter following purification (FIG. 4C), 43.78% of the total protein in the strip fraction was determined to be aggregate, showing that the aggregate preferentially accumulated on the filter.

[0156] Similarly, FIG. 5A shows an analysis of Sample 2 before purification, where aggregate amounted to 11.15% of the total protein content. FIG. 5B provides the results of the purification following elution of the protein preparation through the filter, where, for recovered protein, aggregate now amounted to only 1.11% of the total protein content in Sample 2. Following stripping of the filter following purification (FIG. 5C), 55.43% of the total protein in the strip fraction was determined to be aggregate, showing that theIGM Ref. No. 008-075 WO 1 aggregate preferentially accumulated on the filter. As can be seen, the scale-up from 150 mg total protein to 13.7 g total protein had essentially no discernible effect on the efficiency of the process for removing aggregates from the exemplary imvotamab preparation and illustrates the scalability and efficiency of the methods provided herein.

[0157] The recovered antibody contained only 1.11 - 1.38% aggregates following a single purification run utilizing the methods disclosed herein.EXAMPLE 3POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY ANTI-CD38 x ANTI-CD3 BISPECIFIC PENTAMERIC IGM ANTIBODY

[0158] Two samples were prepared of a bispecific, pentameric IgM antibody with ten binding domains that bind to CD38 and a single anti-CD3 binding domain on the J chain (IGM B-2 as described in International Patent Publication No. WO 2023 / 150677). The first preparation contained 49 mg total protein (Sample 1) and the second preparation contained 330 mg total protein (Sample 2). Each sample, in a NaPi / NaCl solvent mixture, was diluted with a solution containing poly(ethylene glycol) as a displacement enhancer (10 mM NaPi, 175 mM NaCl, 24% (w / v) PEG-4600, pH 7.00) to bring the concentration of PEG 4600 in each sample to 3.00% (w / v) while keeping the NaCl, NaPi, and pH constant. The two sample preparations were each purified using an AKTA PURE™ chromatography system loaded with a 3M EMPHAZE™ AEX filter as the solid phase. Parameters related to the purification of the two sample preparations are provided in Table 3 below.*represents reduction in aggregate mass relative to starting sample. For example, if starting sample contains 10 mg of HMWI, and end sample contains 1 mg of HMWI, HMWI removal efficiency is 90% [100% - (1 mg / 10 mg) = 90% aggregate removal]. Typically, LMW’s are excluded from the integration in order to analyze aggregate removal efficiency by utilizing the HMWI to Target protein ratio.IGM Ref. No. 008-075 WO 1

[0159] FIG. 6A provides a chromatographic analysis of Sample 1 prior to purification, where aggregate content amounted to 7.10% of the total protein in the sample. FIG. 6B provides a chromatographic analysis of Sample 1 following purification via displacement chromatography; as can be seen, following a single purification pass, the amount of aggregates in the sample was reduced to 2.50% of the total protein content. The filter was stripped following purification, and the strip fraction was analyzed as shown in FIG. 6C; chromatographic analysis revealed that 31.34% of the total protein in the strip fraction was aggregate, illustrating that protein aggregates preferentially accumulated on the filter.

[0160] Similarly, FIG. 7A provides a chromatographic analysis of Sample 2 prior to purification, where aggregate amounted to 7.51% of the total protein in the sample. FIG. 7B provides a chromatographic analysis of Sample 2 following purification via displacement chromatography; as can be seen, following a single purification pass, the amount of aggregates in the sample was reduced to 0.47% of the total protein content. The filter was stripped following purification and the strip fraction was analyzed as shown in FIG. 7C; chromatographic analysis revealed that 36.63% of the total protein in the strip fraction was aggregate, showing that the aggregate preferentially accumulated on the filter.

[0161] Scale-up of the process from 49 mg total protein to 330 mg total protein (over 6- fold) did not adversely affect the efficiency of the process for removing aggregate from the anti-CD38xanti-CD3 bispecific IgM antibody preparation and illustrates the applicability of the process to antibody mixtures of different scales.

[0162] The above process has also been carried out successfully on even larger amounts of protein, successfully recovering 800 grams of the anti-CD38xanti-CD3 bispecific, pentameric IgM antibody (data not shown).EXAMPLE 4POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY ANTI-SARS-CoV-2 PENTAMERIC IGM ANTIBODY

[0163] Two samples containing non-aggregated and aggregated anti-SARS-CoV-2 pentameric IgM antibody were purified in accordance with the methods provided herein. The anti-SARS-CoV-2 pentameric IgM antibody contained in the samples is described inIGM Ref. No. 008-075 WO 1 U.S. Patent Application Publication No. US 2022 / 0267415. This antibody has a smaller molecular mass than the antibodies used in Examples 1-3; its molecular mass is 879,711 daltons.

[0164] The first sample preparation (Sample 1) contained 112 mg total protein and the second preparation (Sample 2) contained 162 mg total protein. Each sample, in a NaPi / NaCl solvent mixture, was diluted with a solution containing PEG as a displacement enhancer (10 mM NaPi, 175 mM NaCl, 24% (w / v) PEG-4600, pH 7.00) to bring the concentration of PEG 4600 in the samples to 1.92% (w / v) while keeping the NaCl, NaPi, and pH constant. The two sample preparations were each purified using an AKTA PURE™ chromatography system loaded with a 3M EMPHAZE™ AEX filter. Parameters related to the purification of the two sample preparations are provided in Table 4 below.

[0165] FIG. 8A shows a chromatographic analysis of Sample 1 prior to purification, where aggregates amounted to 11.03% of the total protein contained in the sample. FIG. 8B provides a chromatographic analysis of Sample 1 following purification via displacement chromatography; as can be seen, following a single purification pass, the amount of aggregates in the sample was reduced to 1.77% of the total protein content. The filter was stripped following purification and the strip fraction was analyzed as shown in FIG. 8C; chromatographic analysis revealed that 30.52% of the total protein in the strip fraction was aggregate, showing that the aggregate preferentially accumulated on the filter.

[0166] Similarly, FIG. 9A shows a chromatographic analysis of Sample 2 prior to purification, where aggregates amounted to 2.98% of the total protein contained in the sample. FIG. 9B provides a chromatographic analysis of Sample 2 following purification via displacement chromatography; as can be seen, following a single purification pass, the amount of aggregates in the sample was reduced to 1.18% of the total proteinIGM Ref. No. 008-075 WO 1 content. The filter was stripped following purification and the strip fraction was analyzed as shown in FIG. 9C; chromatographic analysis revealed that 13.02% of the total protein in the strip fraction was aggregate, showing that the aggregate preferentially accumulated on the filter.EXAMPLE 5HOST CELL PROTEIN CLEARANCE USING POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY

[0167] The methods described herein are particularly effective at removing host cell proteins (HCP), among other things, from preparations containing, for example, an IgM antibody. A series of three purification runs similar to FIG. 2 were performed on sample preparations containing, in addition to host cell proteins, the IgM antibody, imvotamab. In each of the runs carried out, the HCP content was assayed at each step of the process. (FIG. 2 presents a non-comprehensive schematic of optional, additional purification steps that can be used in combination with the methods of the present disclosure).

[0168] For Run 1, a 20 mL POROS® HQ 50 anion exchange column, 1.1 cm diameter, 20 cm bed height was used for the capture step. The sample was loaded onto the column at a loading density of 14 mg of IgM antibody per mL of bed volume. The sample was eluted using a gradient starting with 100 mM NaCl, 10 mM NaPi, 3% PEG 4600 w / v, at pH 7.00 to 100% of an eluent containing 1 M NaCl, 10 mM NaPi 3% PEG 4600 w / v, at pH 7.00, fractionating the target imvotamab elution peak. Following the capture step in which the target antibody was collected (i.e., “captured”), the captured antibody was then loaded at 18.8 mg of IgM antibody per mL of bed volume and subjected to a further intermediate purification by passage through a column (1.1 cm diameter, 20 cm bed height) containing an illustrative resin, Capto Core 400 (CC400, Cytiva), in flowthrough mode. For the CC400 flowthrough (FT) step, the salt (NaCl) concentration of the HQ elution was brought to -165 mM NaCl, 10 mM NaPi, at pH 7.00, by diluting the pool with 10 mM NaPi, pH 7.00, 3% PEG 4600 w / v. The CC400 was operated in a FT mode using 165 mM NaCl, 10 mm NaPi, 3% PEG 4600 w / v, pH 7.00. Following the illustrative CC400 intermediate purification step, polymer-assisted displacement chromatography was carried out using a 3M EMPHAZE™ AEX hybrid filter (EMP201 AEX 020R) as previously described. The loading density of the sample wasIGM Ref. No. 008-075 WO 1 approximately 49 mg / mL on 2.8 mL BV (bed volume) EMPHAZE™ hybrid filter (two 1.4 mL BV EMP201 AEX 020R filters connected in series). See FIG. 10A. Additional purification / polishings steps can optionally be carried out, if desired, for example, using a CHT column as indicated in Table 5 below.

[0169] Run 2 was carried out as generally described for Run 1 above, but at a different scale and using a different cell culture lot.

[0170] For Run 3, a 20 mL TMAE (trimethylammoniummethyl) column (1.1 cm diameter, 20 cm bed height) was used to capture the IgM antibody. The column was loaded with harvest cell culture fluid at a loading density of 13 mg of IgM antibody per mL of bed volume. The sample was eluted in a solvent gradient of 100 mM NaCl, 10 mM NaPi, pH 7.00, 3% PEG 4600 w / v to 100% of 1 M NaCl, 10 mM NaPi 3% PEG 4600 w / v, pH 7.00 to thereby fractionate the target imvotamab elution peak. In a subsequent tandem purification step, two CC400 columns, each at 10 cm bed height, 0.8 cm diameter, 5 mL bed volume (CYTIVA Hi Screen™ Capto Core columns) were attached to four 1.4 mL (EMP201AEX 020R) filters. Both the columns and filters were connected in series. TMAE eluate (containing 200 mg of imvotamab + HMWI) that had been diluted to -165 mM NaCl, 10 mM NaPi, 3% PEG 4600 w / v, pH 7.0 was then passed through the tandem chromatography separation steps. Additional purification / polishings steps can optionally be carried out, if desired, e.g., using a CHT or other suitable chromatography media.

[0171] The results of this series of runs are summarized in Table 5 below.IGM Ref. No. 008-075 WO 1

[0172] In all three runs, the displacement chromatography step was effective to achieve a greater reduction in HCP content than in any of the other purification steps, with the reduction in HCP being several-fold greater for the displacement chromatography step in comparison to the other purification steps. See the column labeled, “Fold HCP Reduction” which highlights the superiority of the displacement chromatography step over other chromatographic separations.

[0173] In the third of the three purifications (Run 3), the output from the CC400 step was fed directly through the 3M EMPHAZE filter in a single operation, in contrast to Runs 1 and 2, where the separations were carried out as separate operations. The combination of the two steps proved very effective at HCP clearance, achieving a 733 -fold reduction in HCP relative to the HCP content in the feed input to the tandem chromatography steps.EXAMPLE 6POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION USING A RETROVIRUS AS A DISPLACER MOLECULE

[0174] Examples 1-5 provide examples of purification of high molecular weight molecules, e.g., IgM antibodies, in which aggregated IgM antibody acts as the displacer during displacement chromatographic purification of unaggregated IgM antibodies in the presence of a displacement enhancer molecule such as PEG. HMWI particles other than aggregated protein can also serve as displacers to facilitate purification of a target protein using the methods provided herein.

[0175] In reference to FIGs. 10A-C, displacement chromatography can be carried out using any of a number of displacer molecules such as, for example, a retrovirus. To illustrate this point, chromatographic separations of preparations of imvotamab were carried out in a solution comprising 165 mM NaCl, 10 mM NaPi, 3% PEG 4600 w / v, at pH 7.00.

[0176] In reference to FIG. 10A, the top plot corresponds to Run 1 in Example 5.

[0177] In reference to FIG. 10B (“Run B”), a 20 mL POROS® HQ 50 column, 1.1 cm diameter, 20 cm bed height was used for the capture step. The column was loaded with harvest cell culture fluid at a loading density of 12 mg / mL of imvotamab. The sample was eluted using a mobile phase gradient starting with 100% 100 mM NaCl, 10 mM NaPi, pH 7.00, 3% PEG 4600 w / v (Mobile Phase A) to 75% 100 mM NaCl, 10 mMIGM Ref. No. 008-075 WO 1NaPi, pH 7.00, 3% PEG 4600 w / v (Mobile Phase A) and 25% of 1 M NaCl, 10 mM NaPi, 3% PEG 4600 w / v, pH 7.00 (Mobile Phase B). Following capture of imvotamab, the NaCl concentration of the HQ elution was brought to -165 mM NaCl, 10 mM NaPi, at pH 7.00 by diluting the fractionate with 10 mM NaPi, pH 7.00, 3% PEG 4600 w / v to prepare the CC400 chromatography load. The CC400 step was operated in a flow- through mode using 165 mM NaCl, 10 mm NaPi, 3% PEG 4600 w / v, at pH 7.00 as the eluent. Two CC400 columns (CYTIVA HiScreen™ Capto Core columns) connected in series, each having a 10 cm bed height, 0.8 cm diameter, 5 mL bed volume, were used for this separation step. The columns were loaded with the conductivity adjusted HQ pool at a loading density of 17.4 mg / mL of imvotamab. Following the illustrative CC400 intermediate purification step, displacement chromatography was carried out using two 1.4 mL bed volume 3M EMPHAZE™ AEX hybrid filters (EMP201 AEX 020R) connected in series as previously described; sample was loaded at a load density of 40 mg / mL imvotamab. Endogenous CHO Type-C retrovirus (as an exemplary displacement molecule) was added to the load for displacement chromatography to raise its final concentration to 2.63E+06 viral particles (vp) / mL (small retroviral spike). See FIG. 10B.

[0178] In reference to FIG. 10C (“Run C”), the POROS® HQ 50 chromatography capture and CC40 chromatographic separation steps were performed as described above with slight variations in loading density (11.4 mg / mL load density for the HQ separation step, 18.2 mg / mL load density for the CC400 separation step). Displacement chromatography was also carried out as described above but with a difference in loading virus concentration and a loading density of 35 mg / mL imvotamab. Endogenous CHO Type-C retrovirus was added to the load for displacement chromatography to raise the final concentration to 4.05+06 vp / mL, approximately double the amount of virus in Run B.

[0179] In the strip fractions, the virus was concentrated and isolated from the displaced pool in the same manner as previously described for the IgM aggregate. In Run C, it appears that the virus was able to displace the IgM dimeric aggregate from the filter, as the degree of HMWI removal was decreased from Run B to Run C.

[0180] FIG. 10B provides the results of displacement chromatographic purification of an imvotamab antibody preparation to which a small quantity of retrovirus was added (Run B). Addition of retrovirus did not notably impact the purity of the recovered IgM antibody following displacement chromatography (2.5% aggregate post-purification in FIG. 10A vs 2.3% in FIG. 10B) However, we discovered that the added retrovirus wasIGM Ref. No. 008-075 WO 1 captured on the filter during the displacement chromatography step and could be successfully recovered from the filter post-purification. Addition of a larger quantity of retrovirus to the preparation during displacement chromatographic purification (as shown in FIG. 10C) modestly lessened the purity of the unaggregated IgM antibody ultimately recovered (4.42% aggregate post-purification in FIG. 10C vs. 2.3% in FIG. 10B), however, it was determined that the retrovirus was captured on the filter, indicating the usefulness of the methods of the disclosure to, among other things, achieve virus removal from a biomaterial mixture comprising a target biomaterial such as, e.g., an IgM antibody.EXAMPLE 7POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY ANTI-CD123 x ANTI-CD3e BISPECIFIC PENTAMERIC IGM ANTIBODY

[0181] A sample containing aggregated and non-aggregated forms of a bispecific, pentameric IgM antibody with ten binding domains that bind to CD 123 and a single anti- CD3s binding domain on the J chain (h32716- 1 -1 IgM as described in International Patent Application Publication No. WO 2022 / 178047) was prepared. The sample contained 274 mg total protein (based upon ultraviolet absorbance at 280 nm). The sample, in a NaPi / NaCl solvent mixture, was diluted with a solution containing PEG as a displacement enhancer (10 mM NaPi, 175 mM NaCl, 24% (w / v) PEG-4600, pH 7.00) to bring the concentration of PEG 4600 in the sample to 3.00% (w / v). The preparation was purified as described in Example 2 above using an AKTA PURE™ chromatography system loaded with a 3M EMPHAZE™ AEX filter as the solid phase (chromatography medium). The load density and other parameters of the preparation are provided in Table 6 below.*represents reduction in aggregate mass relative to starting sample. For example, if starting sample contains 10 mg of HMWI, and end sample contains 1 mg of HMWI, HMWI removal efficiency is at 90% [100%- (1 mg / 10IGM Ref. No. 008-075 WO 1 mg)=90% aggregate removal] . Typically, LMW’ s are excluded from the integration in order to analyze aggregate removal efficiency by utilizing the HMWI to Target protein ratio.

[0182] FIG. 11A provides a chromatographic analysis of the preparation prior to purification, illustrating that aggregate amounted to 4.21% of the total protein in the sample. FIG. 11B provides a chromatographic analysis of the purified preparation following displacement chromatography, illustrating a reduction in aggregate amount to only 1.51% of the total protein content in the displaced material. FIG. 11C provides a chromatographic analysis of the strip fraction, illustrating that 17.76% of the total protein in the strip fraction was aggregate, showing that the aggregate preferentially accumulated on the filter.EXAMPLE 8 POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY ANTI-DR5 PENTAMERIC IGM ANTIBODY

[0183] Two sample preparations containing non-aggregated target and aggregates of a pentameric IgM antibody with ten binding domains that bind to DR5, aplitabart (IGM- 8444, Wang, B.T., et al., Mol Cancer Ther. 2021 Dec 1; 20(12): 2483-2494), were prepared. Both preparations contained 132 mg total protein. The first sample, in a NaPi / Tris / NaCl solvent mixture, was diluted with a solution containing PEG as a displacement enhancer (10 mM NaPi, 20 mM Tris, 160 mM NaCl, 24% (w / v) PEG 4600, pH 8.25) to bring the concentration of PEG 4600 in the sample to 1.92% (w / v). The second sample was diluted in a NaPi / Tris / NaCl solvent mixture containing PEG as a displacement enhancer (10 mM NaPi, 20 mm Tris, 165 mM NaCl, 24% (w / v) PEG 4600, pH 8.25) to bring the concentration of PEG 4600 in the sample up to 2.04% (w / v). The two sample preparations were each purified in an AKTA PURE™ chromatography system with a 3M EMPHAZE™ AEX filter as the solid phase. The loading density and other parameters of the two sample preparations are shown in Table 7 below.IGM Ref. No. 008-075 WO 1*represents reduction in aggregate mass relative to starting sample. For example, if starting sample contains 10 mg of HMWI, and end sample contains 1 mg of HMWI, HMWI removal efficiency is at 90% [100%-(l mg / 10 mg)=90% aggregate removal]. Typically, LMW’s are excluded from the integration in order to analyze aggregate removal efficiency by utilizing the HMWI to Target protein ratio.

[0184] FIG. 12A provides a chromatographic analysis of Sample 1 prior to purification, where aggregate amounted to 6.74% of the total protein in the sample. FIG. 12B provides a chromatographic analysis of the purified preparation following displacement chromatography, illustrating a reduction in aggregate to 2.32% of the total protein content. Following stripping of the filter following purification (FIG. 12C), chromatographic analysis of the strip fraction revealed that 29.72% of the total protein was aggregate, again illustrating that the aggregate preferentially accumulated on the filter. These results further illustrate the capability of the methods provided herein to provide highly purified antibody or other preparations having low levels of protein aggregates.

[0185] Similarly, FIG. 13A provides a chromatographic analysis of Sample 2 prior to purification, where aggregate amounted to 2.90% of the total protein in the sample. FIG. 13B provides a chromatographic analysis of the purified preparation following displacement chromatography, illustrating a reduction in aggregate amount to only 1.20% of the total protein content. Following stripping of the filter following purification (FIG. 13C), chromatographic analysis revealed that 17.13% of the total protein in the strip fraction was aggregate, illustrating that the aggregate preferentially accumulated on the filter.EXAMPLE 9POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY PURIFICATION OF AN EXEMPLARY ANTI-DR5 PENTAMERIC IGM ANTIBODY: EVALUATION OF VIRAL CLEARANCE

[0186] As illustrated herein, we have discovered that displacement chromatography can be employed to remove viruses, e.g., by utilizing a virus to be removed as a displacer molecule. To illustrate this point, chromatographic separations of preparations containing an anti-DR5 pentameric IgM antibody and an exemplary virus, e.g., Minute Virus of Mice (MVM) or Xenotropic Murine Leukemia Virus (xMuLV), were carried out using both conventional bind and elute chromatographic methods as well as the polymer-IGM Ref. No. 008-075 WO 1 assisted displacement chromatography method provided herein to highlight the differences in the two methods.

[0187] Samples contained a pentameric IgM antibody with ten identical binding domains that bind to DR5, each comprising the VH and VL amino acid sequences, SEQ ID NO: 5 and SEQ ID NO:6, respectively, described in U.S. Patent No. 9,938,347. The samples were subjected to both conventional bind and elute chromatography as well as polymer- assisted displacement chromatography as described previously to explore the efficiency of virus removal.

[0188] More particularly, the IgM antibody preparations were subjected to various upstream treatment and / or chromatographic purification steps (details not provided) including detergent treatment (60 minutes), TMAE anion exchange chromatography, Capto™ MMC (multimodal chromatography, CYTIVA), and CHT (ceramic hydroxyapatite) TYPE II chromatography, as shown in Tables 8 and 9. For the displacement chromatography, an aqueous solution containing 24% w / v PEG-4600, 10 mM NaPi, at pH 7.50 was added to the intermediate Capto™ MMC product to bring the PEG 4600 concentration to 3% w / v, followed by addition of an aqueous solution containing PEG 4600 diluent (3% w / v PEG 4600, 10 mM NaPi, pH 7.00) to the pool to bring the NaCl concentration to 165 mM NaCl. Four 1.4 mL bed volume 3M EMPHAZE™ AEX hybrid filters (EMP201 AEX 020R) as previously described were cleaned with 1 M NaOH, followed by passage of 500 mM NaPi at pH 7.00 through the filters to neutralize the sodium hydroxide. The filters were then equilibrated with a solution containing 165 mM NaCl, 10 mM NaPi, 3% PEG 4600 w / v, pH 7.00. Duplicate runs for each exemplary virus (MVM and xMuLV) were executed to test the viral clearance capacity of 3M EMPHAZE™ AEX hybrid filters operated in the displacement chromatography mode provided herein, using an exemplary enhancement displacer, i.e., poly(ethylene glycol). The large retrovirus, xMuLV, was evaluated at a virus spike mass of 7.65 logs of PFU (plaque-forming units). The small virus, MVM, was also evaluated at a virus spike mass of 7.65 logs of PFU. All replicates had an IgM antibody load density of 135 mg of IgM antibody per mL of bed volume. Results are provided in Tables 8 and 9.Table 8. Clearance of xMuLVXMuLVIGM Ref. No. 008-075 WO 1Table 9. Clearance of MVM

[0189] As can be seen from the results in Table 8 above, the displacement enhancer- assisted chromatography method provided herein is extremely effective at the removal of5 large viruses such as xMuLV, as illustrated by the notable log reduction values shown above, in particular, when compared to other purification or chromatographic separation methods such as bind and elute (B / E).

[0190] While the displacement enhancer-assisted chromatography method provided herein, based upon the results provided above, appears to be less effective for small virus10 removal than for large virus removal, e.g., from a sample of a protein such as the exemplary anti-DR5 pentameric IgM antibody used in this example, chromatographic separation conditions can likely be modulated to more effectively remove virus, e.g., based upon the relative sizes of the virus and the target molecule (in both aggregated andIGM Ref. No. 008-075 WO 1 non-aggregated form). For example, in considering an IgG antibody, which is generally smaller than an IgM, the MVM or other similarly-sized virus should outcompete the IgG during the displacement chromatography step and be removed to a large degree, if not totally, from solution.

[0191] Considering the results in Tables 8 and 9 above, it can be seen that a large difference in virus clearance is observed when comparing the anion exchange bind and elute chromatographic separation (e.g., AEX BZE) and the polymer-assisted displacement chromatography using an anion exchange medium. In considering the results in Table 9, the AEX BZE chromatography resulted in removal of MVM, while the AEX polymer- assisted displacement chromatography was approximately ten times worse / ineffective at removing a virus that is smaller than the target protein. In contrast, polymer-assisted displacement chromatography was 1000 times more effective at removing a large virus such as MXuLV (i.e., a virus that is larger than the target protein) than AEX BZE. The foregoing demonstrates the fundamental chemical differences between the different chromatographic approaches (i.e., bind and elute chromatography versus displacement chromatography) despite both employing an anion exchange medium. Generally, AEX polymer-assisted displacement chromatography can be considered to be a chemical based sized exclusion method using AEX media as a convenient, but not necessarily the only, substrate, while AEX BZE is a traditional electrostatic-based chromatographic separation method.

[0192] As can be seen from the data provided above, the displacement chromatography methods provided herein can be employed to achieve highly purified large biologies.EXAMPLE 10POLYMER-ASSISTED DISPACEMENT CHROMATOGRAPHY USING A CHT CHROMATOGRAPHY MEDIUM PURIFICATION OF AN EXEMPLARY ANTI-DR5 PENTAMERIC IGM ANTIBODY

[0193] A sample preparation containing a target biomaterial mixture comprising a pentameric IgM antibody with ten binding domains that bind to DR5, aplitabart, and aplitabart aggregates, was prepared. The sample contained 69 mg total protein. The sample was diluted with an aqueous NaPi / Tris / NaCl solvent mixture containing PEG- 6000 as a displacement enhancer to bring the concentration of PEG-6000 in the sample to 1.68% (w / v). The estimated composition of the sample was 35-40 mM NaPi, 250-300IGM Ref. No. 008-075 WO 1 mM NaCl, 15-20 mM Tris Cl, 2-5 mM NaOAc, 1.68% PEG-6000 w / v, at pH 7.00. The preparation was purified using a 1 mL total volume CHT Type II column as the stationary phase, with continuous loading of the aqueous loading solution onto the CHT column. Eluted samples were analyzed; results are provided in Table 10 below, where sample designations (A7, A8, Bl, etc.) represent the collected fractions. The samples listed in column 1 show samples collected over time from the start of sample loading. Table 10. Purification of an Exemplary Pentameric IgM antibody Targeting DR5*represents percentage reduction in aggregate relative to preceding sample.IGM Ref. No. 008-075 WO 1

[0194] FIG. 14 provides a chromatographic analysis of the biomaterial mixture over the course of displacement chromatography purification, where aggregate amounted to 10.1% of the total protein in the sample prior to purification. FIG. 14B shows the biomaterial mixture that is loaded on the column (“starting material”). FIG. 14C provides a chromatographic analysis of the purified preparation at the beginning of displacement chromatography, illustrating a reduction in aggregate to 2.0% of the total protein content. FIG. 14D provides a chromatographic analysis of the purified preparation at the end of sample loading during displacement chromatography, illustrating a reduction in aggregate to 5.8% of the total protein content. FIG. 14E provides a chromatographic analysis of the purified preparation at the end of displacement chromatography, illustrating a reduction in aggregate to 1.8% of the total protein content.

[0195] A loss in aggregate removal efficiency towards the end of sample loading during polymer-assisted displacement chromatography is often observed (sample B1-B4), as is the ‘tailing’ behavior seen in Fig 14E. The calculated cumulative effect of an unoptimized CHT Il-mixed modal displacement chromatographic purification utilizing PEG is summarized in Table 10, illustrating a cumulative aggregate removal of 59.21% from the starting aggregate level of 10.10% as seen in FIG 14B.

Claims

IGM Ref. No. 008-075 WO 1WHAT IS CLAIMED IS1. A method for separating components of a biomaterial mixture, the method comprising:(a) applying an aqueous loading solution comprising the biomaterial mixture, a salt, and 0.1% (w / v) to 10% (w / v) of a non-naturally occurring, non-ionic, water- soluble polymer to a chromatography medium to thereby initially bind components of the biomaterial mixture to the chromatography medium, wherein the non-naturally occurring water-soluble polymer has an average molecular weight from about 300 to about 100,000 daltons and the biomaterial mixture comprises a high molecular weight biomaterial impurity (HMWI) and a target biomaterial having a lower molecular weight than the HMWI; and(b) continuously applying the loading solution to the chromatography medium to effect displacement of the target biomaterial from the chromatography medium, thereby providing an eluate comprising the target biomaterial at a higher purity relative to that of the loading solution.

2. The method of claim 1, wherein step (b) is carried out in the absence of an elution gradient.

393. The method of claim 1 or claim 2, wherein the biomaterial mixture comprised in the loading solution contains from about 20 percent to about 1 percent HMWI of total biomaterial in the mixture, from about 18 percent to about 3 percent HMWI of total biomaterial in the mixture, or from about 15 percent to about 7 percent HMWI of total biomaterial in the mixture, inclusive of all ranges falling between members of each of the foregoing.

4. The method of any one of claims 1 to 3, wherein the biomaterial mixture comprises at least one of a protein, peptide, virus, liposome, nucleic acid.

5. The method of any one of claims 1 to 4, wherein the HMWI is an aggregated protein, a host-cell protein, a virus, a nucleic acid, or a combination thereof.

6. The method of any one of claims 1-5, wherein the target biomaterial is a protein and the HMWI is comprises at least one of an aggregated protein, host-cell protein, virus or a combination thereof.IGM Ref. No. 008-075 WO 17. The method of any one of claims 1-6, wherein the target biomaterial is an antibody or an antibody fragment.

8. The method of claim 7, wherein the antibody is an IgA or an IgM antibody.

9. The method of any one of claims 1-8, wherein the non-naturally occurring, non-ionic water-soluble polymer is a poly(ethylene glycol) (PEG), a polyacrylamide (PAM), a polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or a copolymer or combination thereof.

10. The method of claim 9, wherein the non-naturally occurring, non-ionic, water- soluble polymer is a poly(ethylene glycol) (PEG).

11. The method of any one of the foregoing claims, wherein the non-naturally occurring, non-ionic water-soluble polymer has an average molecular weight between about 300 daltons and about 40,000 daltons, between about 350 daltons and about 30,000 daltons, between about 350 daltons and about 20,000 daltons, between about 350 daltons, and about 10,000 daltons, between about 350 daltons and about 6,000 daltons, and between about 400 daltons and about 5,000 daltons, inclusive of all ranges falling between members of each of the foregoing.

12. The method of any one of the foregoing claims, wherein the loading solution further comprises a buffer.

13. The method of claim 12, wherein the buffer comprises a phosphate buffer, a Good’s buffer, an amino acid buffer, or a combination of one or more of the foregoing.

14. The method of any one of the foregoing claims, wherein the pH of the loading solution ranges from about 5.0 to about 8.6.

15. The method of any one of the foregoing claims, where loading solution comprises from about 0.10 percent to about 10 percent (w / v) of the non-naturally occurring, non-ionic, water-soluble polymer, or from about 0.50 percent to about 5.0 percent (w / v) of the non-naturally occurring, non-ionic, water-soluble polymer.

16. The method of any one of the foregoing claims, wherein the salt comprised in the loading solution is sodium chloride, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, tris chloride or a combination of one or more of the foregoing.

17. The method of any one of the foregoing claims, wherein the loading solution comprises a salt concentration from about 75 mM to about 500 mM.

18. The method of any one or more of the foregoing claims, wherein the loading solution is applied to the chromatography medium at a loading density of from about 15IGM Ref. No. 008-075 WO 1 mg / mL to about 350 mg / mL target biomaterial, from about 20 mg / mL to about 300 mg / mL target biomaterial, from about 25 mg / mL to about 200 mg / mL target biomaterial, from about 25 mg / mL to about 100 mg / mL target biomaterial, from about 20 mg / mL to about 80 mg / mL target biomaterial, or from about 20 mg / mL to 50 mg / mL target biomaterial.

19. The method of any one or more of the foregoing claims, wherein the chromatography medium is an ion exchange medium, a metal affinity medium, or a mixedmode medium.

20. The method of claim 19, wherein the chromatography medium is an ion exchange medium.

21. The method of claim 20, wherein the chromatography medium is an anion exchange medium.

22. The method of claim 20, wherein the chromatography medium is a cation exchange medium.

23. The method of claim 20, wherein the chromatography medium is a mixed mode medium.

24. The method of any one of the foregoing claims, wherein the chromatography medium is housed in a column.

25. The method of any one of claims 1-23, wherein the chromatography medium is comprised in a filter.

26. The method of any one of the foregoing claims, wherein the HMWI comprises a host cell protein, and the host cell protein content of the loading solution does not exceed 50,000 parts per million, does not exceed 10,000 parts per million, or does not exceed 4,000 parts per million.

27. The method of any one of the foregoing claims, wherein the target biomaterial is a multimeric binding molecule.

28. The method of claim 27, wherein the multimeric binding molecule is a multimeric antibody comprising two, four, five, or six bivalent binding units, and wherein each binding unit comprises two heavy chains each comprising at least the heavy chain variable region of a binding domain and an IgA heavy chain constant region or multimerizing fragment or variant thereof or an IgM heavy chain constant region or multimerizing fragment or variant thereof.

29. The method of claim 28, wherein the multimeric antibody is a pentameric antibody.IGM Ref. No. 008-075 WO 130. The method of any one of the foregoing claims, wherein the target biomaterial is an antibody as described in the accompanying disclosure.

31. The method of any one of the foregoing claims, further comprising (c) recovering the target biomaterial displaced from the chromatography medium as a recovered target biomaterial.

32. The method of claim 31, effective to remove 65 percent or more, 70 percent or more, 75 percent or more, 80 percent or more, or 90 percent or more, HMWI from the biomaterial mixture in step (a).

33. The method of claim 31 or claim 32, wherein the recovered target biomaterial contains less than 5 percent, less than 4 percent, less than 3 percent, or less than 2 percent HMWI.

34. The method of claim 32, wherein the recovered target biomaterial contains no more than 0.25 percent HMWI.

35. The method of any one of claims 1-25 or 27-34, wherein the HMWI comprises a virus, and the method results in from 3 to 7 logs of virus clearance from the biomaterial mixture.

36. The method of any one of the foregoing claims, wherein the biomaterial mixture has undergone one or more pre-purification steps.

37. The method of claim 36, wherein the one or more pre-purification steps include a flow-through purification.

38. The method of claim 36, wherein the one or more flow-through prepurification steps include a capture step and / or one or more polishing steps.

39. The method of any one of the foregoing claims, further comprising, prior to step (a), and with respect to any one of claims 36-38, following the one or more prepurification steps, flushing the chromatography medium with an aqueous buffer.

40. The method of any one of the foregoing claims, further comprising, prior to applying step (a), and with respect to any one of claims 36-39, following the one or more prepurification steps, washing the chromatography medium with an aqueous equilibration buffer having the same components and concentrations of components as the loading solution absent the biomaterial mixture.

41. The method of claim 40, wherein the chromatography medium is an anion exchange medium, the loading solution comprises sodium chloride at a concentration ranging from about 75 mM to 400 mM (e.g., 165 mM, or 210 mM), sodium phosphate at aIGM Ref. No. 008-075 WO 1 concentration ranging from about 1 mM to about 25 mM (e.g., lOmM), and from about 0.5 to about 7 percent (w / v) poly(ethylene glycol) (e.g., about 2 percent (w / v)) having an average molecular weight from about 1000 to about 10,000 (e.g., 4600) daltons.

42. The method of claim 40 or claim 41, wherein the recovered target biomaterial contains less than 4 percent HMWI, less than 3 percent HMWI, or less than 2 percent HMWI.

43. The method of any one of claims 31 to 42, further comprising (d) isolating the recovered target material.

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