Methods of reducing the rate of enzymatic hydrolysis activity in compositions obtained from purification platforms

Abstract

The present invention relates to methods of reducing the rate of enzymatic hydrolysis activity in compositions obtained from a purification platform, and provides purification platforms comprising a depth filtration step and / or a hydrophobic interaction chromatography (HIC) step. The present invention also discloses methods of using the purification platforms described herein and compositions, such as pharmaceutical compositions, obtained from the purification platforms.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080033105.0, filed on May 1, 2020, entitled "Method for reducing the rate of enzyme hydrolysis activity in a composition obtained from a purification platform".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 961,609, filed January 15, 2020, and U.S. Provisional Patent Application No. 62 / 843,261, filed May 3, 2019, the disclosures of which are hereby incorporated in their entirety by reference. Technical Field

[0004] This disclosure provides a purification platform that includes a depth filtration step and / or a hydrophobic interaction chromatography (HIC) step. Methods using the purification platform described herein and compositions obtained from said purification platform are also disclosed herein. Background Technology

[0005] Biotherapeutic products derived from host cell cultures (such as antibodies) require purification to remove host cell proteins and other impurities that may affect, for example, product quality and therapeutic efficacy. Current purification methods may not remove all host cell proteins and impurities, including host cell hydrolases. Therefore, residual host cell proteins and impurities along with the purification target can affect the purification target itself, as well as other additives, such as formulation components like surfactants. Thus, there is a need for improved methods for purifying biotherapeutic products derived from host cell cultures for pharmaceutical use.

[0006] All references cited in this article (including patent applications and publications) are incorporated in their entirety. Summary of the Invention

[0007] In one aspect, a method is provided for reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform including: (a) a capture step; and (b) a depth filtration step, thereby reducing the rate of enzymatic hydrolysis activity of the composition compared to purifying the sample using the same purification platform without the depth filtration step.

[0008] In some embodiments, the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate. In some embodiments, the relative reduction in the enzyme hydrolysis activity rate of the composition is at least about 20% compared to purifying the sample using the same purification platform without a depth filtration step.

[0009] On the other hand, a method is provided for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform including: (a) a capture step; and (b) a deep filtration step, thereby reducing the level of the hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the deep filtration step. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate. In some embodiments, the relative reduction in the level of one or more hydrolytic enzymes in the composition is at least about 20% compared to purifying the sample using the same purification platform without the deep filtration step.

[0010] On the other hand, a method is provided for reducing the degradation of polysorbate esters in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform including: (a) a capture step; and (b) a depth filtration step, thereby reducing the degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without the depth filtration step. In some embodiments, the relative reduction in the degradation of polysorbate esters in the composition is at least about 5% compared to purifying the sample using the same purification platform without the depth filtration step.

[0011] In some embodiments, the purification platform is used to purify a target from a sample, wherein the sample contains the target and one or more host cell impurities. In some embodiments, the target contains a peptide. In some embodiments, the host cell impurity is a host cell protein.

[0012] In some embodiments, the depth filtering step is performed before the capture step, or the depth filtering step is performed after the capture step.

[0013] In some embodiments, the depth filtration step includes treatment through a depth filter. In some embodiments, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a viscous resin, and an ash composition. In some embodiments, at least a portion of the substrate of the depth filter includes surface modification. In some embodiments, the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification, and anionic surface modification. In some embodiments, the depth filter is selected from the group consisting of EMPHAZE™ depth filters, XOSP depth filters, PDD1 depth filters, ZETA PLUS™ 120ZA depth filters, and ZETA PLUS™ 120ZB depth filters.

[0014] In some embodiments, the capture step includes processing by affinity chromatography. In some embodiments, affinity chromatography is selected from the group consisting of protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, FcXL chromatography, protein XL chromatography, κ chromatography, and κXL chromatography.

[0015] In some embodiments, the purification platform further includes a virus inactivation step, wherein the virus inactivation step is performed after the capture step. In some embodiments, a deep filtering step is performed after the virus inactivation step.

[0016] In some embodiments, the purification platform further includes another depth filtration step performed prior to the capture step.

[0017] In some embodiments, the purification platform further includes one or more purification steps, wherein the one or more purification steps are performed after a capture step, a deep filtration step, and, if present, a virus inactivation step. In some embodiments, the one or more purification steps include a peptide purification step. In some embodiments, the purification platform further includes another deep filtration step performed before, between, or after the one or more purification steps.

[0018] In some embodiments, the purification platform further includes an ultrafiltration / percolation (UFDF) step, wherein the UFDF step is performed after the one or more purification steps. In some embodiments, the purification platform further includes another depth filtration step performed before or after the UFDF step.

[0019] In some embodiments, the purification platform further includes a hydrophobic interaction chromatography (HIC) purification step. In some embodiments, the HIC purification step is performed before, between, or after the one or more purification steps, if present. In some embodiments, the HIC purification step is performed after the one or more purification steps and before a UFDF step, if present.

[0020] In some embodiments, the purification platform further includes a pH holding step, wherein the pH holding step is performed after the one or more purification steps, if present, and before the UFDF step.

[0021] In some embodiments, the purification platform further includes a virus filtration step, wherein the virus filtration step is performed after the pH maintenance step and before the UFDF step. In some embodiments, the virus filtration step includes treatment through a virus filter.

[0022] In some embodiments, the HIC purification step includes processing through a HIC filter.

[0023] In some embodiments, the one or more purification steps each independently include treatment by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induced chromatography, ceramic hydroxyapatite chromatography, and multi-component chromatography. In some embodiments, the one or more purification steps each independently include treatment by chromatography selected from the group consisting of: DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel™, Capto MMC, and Capto Adhere.

[0024] On the other hand, a method is provided for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform in the following order: (a) a capture step comprising treatment by affinity chromatography; (b) a virus inactivation step; (c) a second peptide purification step; (d) a third peptide purification step; and (e) an ultrafiltration / percolation (UFDF) step, wherein the purification platform further comprises a deep filtration step performed at one or more of the following times: (i) before the capture step; (ii) after the capture step and before the virus inactivation step; (iii) after the virus inactivation step and before the second peptide purification step; (iv) after the second peptide purification step and before the third peptide purification step; or (v) after the third peptide purification step and before the ultrafiltration / percolation (UFDF) step; thereby reducing the rate of enzymatic hydrolysis of the composition compared to purifying the sample using the same purification platform without the deep filtration step.

[0025] In some embodiments, the purification platform further includes, in the following order: a pH maintenance step and a virus filtration step, performed after the third peptide purification step and before the UFDF step. In some embodiments, the virus filtration step includes treatment through a virus filter.

[0026] In some embodiments, the purification platform further includes a hydrophobic interaction chromatography (HIC) purification step performed at one or more of the following times: (i) after the third peptide purification step and before the pH holding step; (ii) after the pH holding step and before the virus filtration step; or (iii) after the virus filtration step and before the UFDF step.

[0027] In some embodiments, the method further includes determining the rate of enzymatic hydrolysis activity of the composition.

[0028] In some embodiments, the method further includes determining the level of one or more hydrolytic enzymes in the composition.

[0029] In some embodiments, the composition comprises polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0030] In some embodiments, the method further includes a sample processing step.

[0031] In some embodiments, the sample is or is derived from a cell culture sample. In some embodiments, the cell culture sample comprises host cells, wherein the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells. In some embodiments, the sample comprises host cells or components derived from the host cells. In some embodiments, the sample comprises one or more host cell proteins, wherein one or more of the host cell proteins is a hydrolytic enzyme.

[0032] In some embodiments, the hydrolytic enzyme is a lipase, esterase, thioesterase, phospholipase, or ceramide enzyme.

[0033] In some embodiments, the sample contains a target, and the target is an antibody portion. In some embodiments, the antibody portion is a monoclonal antibody. In some embodiments, the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0034] In some embodiments, the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0035] In some embodiments, the antibody portion is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0036] In another aspect, a pharmaceutical composition obtained from any of the methods described herein is provided.

[0037] On the other hand, an antibody fraction composition comprising polysorbate is provided, wherein the composition has a reduced polysorbate hydrolysis activity rate, and wherein the composition has a shelf life of more than 24 months.

[0038] On the other hand, an antibody portion composition comprising an antibody portion and a polysorbate is provided, wherein the composition has a reduced polysorbate hydrolysis activity rate, wherein the shelf life of the composition is extended compared to the shelf life specified in a document submitted to a health authority relating to the formulated antibody portion composition, and wherein the shelf life is extended by at least 6 months compared to the shelf life specified in the document.

[0039] On the other hand, a formulated antibody portion composition comprising an antibody portion is provided, wherein the formulated antibody portion composition has reduced polysorbate degradation, wherein the degradation is reduced by at least about 20% compared to the degradation specified in documents submitted to health authorities relating to the formulated antibody portion composition.

[0040] On the other hand, an antibody portion composition comprising an antibody portion and a polysorbate is provided, wherein the polysorbate degrades by 20% or less per year during storage of the liquid composition.

[0041] In some embodiments, the antibody portion of the formulated antibody partial composition is a monoclonal antibody. In some embodiments, the antibody portion of the formulated antibody partial composition is a human antibody, a humanized antibody, or a chimeric antibody.

[0042] In some embodiments, the antibody portion of the formulated antibody portion composition is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0043] In some embodiments, the antibody portion of the formulated antibody portion composition is selected from the group consisting of: olizumab, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunitozumab, tirelumumab, bevacizumab, rituximab, atezolizumab, olizumab, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinzumab, RO6874281, and RO7122290.

[0044] In some embodiments, the rate of hydrolysis of the polysorbate in the formulated antibody fraction composition is reduced by at least about 20%. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0045] On the other hand, a method is provided for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to a purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the rate of enzymatic hydrolysis of the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps. In some embodiments, the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate. In some embodiments, the relative reduction in the enzyme hydrolysis activity rate of the composition is at least about 20% compared to purifying the sample using the same purification platform without a depth filtration step.

[0046] On the other hand, a method is provided for reducing the level of one or more hydrolases in a composition obtained from a purification platform, the method comprising subjecting a sample to a purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multivariate chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step and before the purification step; or after the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the level of one or more hydrolases in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate. In some embodiments, the relative level of one or more hydrolytic enzymes in the composition is reduced by at least about 20% compared to purifying a sample using the same purification platform without a depth filtration step.

[0047] On the other hand, a method is provided for reducing the degradation of polysorbate esters in a composition obtained from a purification platform, the method comprising subjecting a sample to a purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps. In some embodiments, the relative reduction in degradation of polysorbate in the composition is at least about 5% compared to purifying the sample using the same purification platform without a depth filtration step.

[0048] In some embodiments, a depth filter comprising silica and polyacrylic fibers includes a silica filter aid and polyacrylic fiber pulp.

[0049] In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane includes four layers comprising a hydrogel Q-functionalized nonwoven material and a nine-zone microporous membrane.

[0050] In some embodiments, a depth filter comprising cellulose fibers, diatomaceous earth, and perlite comprises two layers, each layer comprising a cellulose filter matrix impregnated with a filter aid comprising one or more of diatomaceous earth or perlite, and each layer further comprising a resin binder.

[0051] In some embodiments, the depth filter is selected based on the pH of the solution entering the depth filter. In some embodiments, when the pH of the solution entering the depth filter is from about 5 to about 6.5, a depth filter comprising silica and polyacrylic fibers is selected. In some embodiments, when the pH of the solution entering the depth filter is from about 7 to about 8.5, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane is selected. In some embodiments, the method further includes selecting the depth filter based on the pH of the solution entering the depth filter.

[0052] In some embodiments, the purification platform sequentially includes: a deep filtration step, which includes treatment through a deep filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; a capture step, which includes treatment by protein A chromatography; and a purification step.

[0053] In some embodiments, the purification step includes treatment with HIC. In some embodiments, HIC is phenyl SEPHAROSE® rapid flow chromatography.

[0054] In some embodiments, the purification step includes treatment by cation exchange chromatography. In some embodiments, the cation exchange chromatography is POROS® 50HS.

[0055] In some embodiments, the purification platform further includes a second depth filtration step, which includes treatment through a depth filter comprising silica and polyacrylate fibers, wherein the second depth filtration step occurs after the capture step and before the purification step.

[0056] In some embodiments, the purification step includes processing by multivariate chromatography. In some embodiments, the multivariate chromatography is Capto Adhere.

[0057] In some embodiments, the purification platform further includes a second depth filtration step, which includes treatment through a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane, wherein the second depth filtration step occurs after the capture step and before the purification step.

[0058] In some embodiments, the purification platform is used to purify a target from a sample, wherein the sample contains the target and one or more host cell impurities. In some embodiments, the target contains a peptide. In some embodiments, the host cell impurity is a host cell protein.

[0059] In some embodiments, the purification platform further includes a virus inactivation step, wherein the virus inactivation step is performed after the capture step. In some embodiments, the one or more deep filtering steps are performed after the virus inactivation step.

[0060] In some embodiments, the purification platform further includes an ultrafiltration / percolation (UFDF) step, wherein the UFDF step is performed after the purification step.

[0061] In some embodiments, the method described herein further includes determining the rate of enzymatic hydrolysis activity of the composition.

[0062] In some embodiments, the method described herein further includes determining the level of one or more hydrolytic enzymes in the composition.

[0063] In some embodiments, the composition comprises polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0064] In some embodiments, the method described herein further includes a sample processing step.

[0065] In some embodiments, the sample is or is derived from a cell culture sample. In some embodiments, the cell culture sample comprises host cells, wherein the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells. In some embodiments, the sample comprises host cells or components derived from the host cells.

[0066] In some embodiments, the sample comprises one or more host cell proteins, wherein one or more of the host cell proteins is a hydrolase. In some embodiments, the hydrolase is a lipase, esterase, thioesterase, phospholipase, or ceramidinase. In some embodiments, the sample comprises a target, wherein the target is an antibody moiety. In some embodiments, the antibody moiety is a monoclonal antibody. In some embodiments, the antibody moiety is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody. In some embodiments, the antibody portion is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0067] On the other hand, a pharmaceutical composition obtained from any of the methods described herein is provided.

[0068] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. This disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of this disclosure to the specific procedures described herein. Attached Figure Description

[0069] Figure 1A illustrates exemplary steps of the purification platform 100. Figure 1B illustrates exemplary options for the steps of the purification platform.

[0070] Figure 2 shows a bar graph of the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS.

[0071] Figure 3 shows a bar graph of hydrolytic activity measured in the composition obtained from the purification platform using lipase activity assay.

[0072] Figure 4 shows a bar graph of the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS.

[0073] Figure 5 shows a schematic diagram of the purification options for faliximab.

[0074] Figures 6A and 6B show bar graphs of PS20 hydrolytic activity measured in the FcXL eluent of faliximab before (Figure 6A) and after (Figure 6B) filtration with a PDD1 filter.

[0075] Figure 7 shows a bar graph of the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS.

[0076] Figures 8A and 8B show bar graphs of hydrolytic activity measured in the protein A eluate after filtration using a depth filter.

[0077] Figure 9 shows the relative levels of CHOP and polysorbate degradation activity in the protein A eluate after clarification by the EMPHAZE™ depth filter.

[0078] Figure 10 shows the specific activity of the composition obtained from the X0SP depth filter (pH 5.5) for polysorbate degradation at different fluxes.

[0079] Figures 11A to 11C show the specific activity of ozoglucomancil at pH 5.5 (Figure 11A), celumumab at pH 5.5 (Figure 11B), and tocilizumab at pH 6.5 (Figure 11C) for polysorbate degradation at different fluxes.

[0080] Figure 12 shows a bar graph of specific FAMS rates of the composition obtained from the purification platform.

[0081] Figure 13 shows a bar graph of specific FAMS rates of the composition obtained from the purification platform.

[0082] Figure 14 shows a bar graph of specific FAMS rates of the composition obtained from the purification platform.

[0083] Figure 15 shows a bar graph of a specific LEAP rate of the composition obtained from the purification platform.

[0084] Figure 16 shows a schematic diagram of the purification workflow.

[0085] Figure 17 shows a bar graph of the average conversion of the composition obtained from the purification platform, as measured using the LEAP assay.

[0086] Figures 18A and 18B show bar graphs of the hydrolytic activity of the compositions obtained from the purification platform, measured using a lipase activity assay. Figure 18A shows the results obtained from CF 238. Figure 18B shows the results obtained from CF 239.

[0087] Figure 19A shows a bar graph of the average conversion of the composition obtained from the purification platform as measured using the LEAP assay. Figure 19B shows a bar graph of the hydrolytic activity of the composition obtained from the purification platform as measured using the lipase activity assay. Detailed Implementation

[0088] In some aspects, this application provides a method for purifying a target from a sample containing the target, the method comprising subjecting the sample to a purification platform disclosed herein, the purification platform including one or more depth filtration steps and / or one or more hydrophobic interaction chromatography (HIC) steps.

[0089] This disclosure is partly based on the unexpected discovery that purification platforms including one or more deep filtration steps (such as deep filtration of host cell culture medium (HCCF) and / or affinity chromatography eluates) reduce the rate of enzymatic hydrolysis activity of the compositions obtained therefrom. Furthermore, this disclosure is partly based on the unexpected discovery that purification platforms including one or more HIC steps reduce the rate of enzymatic hydrolysis activity of the compositions obtained therefrom, and that purification platforms including both deep filtration and HIC steps can further reduce the rate of enzymatic hydrolysis activity of the compositions obtained therefrom.

[0090] Those skilled in the art will also understand that changes may be made to the form and details of the embodiments described herein without departing from the scope of this disclosure. Furthermore, while various advantages, aspects, and objectives have been described with reference to various embodiments, the scope of this disclosure should not be limited by these advantages, aspects, and objectives.

[0091] definition

[0092] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any definition set forth below and any reference incorporated herein by reference, the definition set forth herein shall prevail.

[0093] The term "antibody moiety" includes a full-length antibody and its antigen-binding fragment. In some embodiments, the full-length antibody comprises two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody and antigen-binding fragments disclosed herein can be defined or identified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDRs of the heavy or light chain are inserted between flanking extensions called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and µ heavy chains, respectively. Several main antibody classes are subclassed, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain). In some embodiments, the antibody portion is a chimeric antibody. In some embodiments, the antibody portion is a semi-synthetic antibody. In some embodiments, the antibody portion is a biantibody. In some embodiments, the antibody portion is a humanized antibody. In some embodiments, the antibody portion is a multispecific antibody, such as a bispecific antibody. In some embodiments, the antibody portion is linked to a fusion protein. In some embodiments, the antibody portion is linked to an immunostimulatory protein, such as interleukin. In some embodiments, the antibody portion is linked to a protein that facilitates entry across the blood-brain barrier.

[0094] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camel-derived single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment bound to an antigen but not containing a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen bound to a parent antibody or a parent antibody fragment (e.g., a parental scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a specific human antibody grafted into a frame region from one or more different human antibodies.

[0095] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, and fragments of these antibodies, provided they exhibit the biological activity of the present invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0096] As used herein, the term "multispecific antibody" refers to a monoclonal antibody that has binding specificity to at least two distinct sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some respects, multispecific antibodies have two binding specificities (bispecific antibodies). In other respects, multispecific antibodies have three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0097] The term "semi-synthetic" in relation to antibodies or antibody moieties refers to an antibody or antibody moieties having one or more naturally occurring sequences and one or more non-natural (i.e., synthetic) sequences.

[0098] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a tightly bound, non-covalently linked dimer of a heavy chain variable region domain and a light chain variable region domain. The folding of these two domains generates six hypervariable rings (three from the heavy chain and three from the light chain), which contribute amino acid residues to achieve antigen binding and impart antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although with lower affinity than the complete binding site.

[0099] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired antigen-binding structure. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0100] The term "biantibody" refers to a small antibody fragment prepared by constructing an scFv fragment (see previous paragraph), in which a short linker (such as about 5 to about 10 residues) is typically present between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific biantibody is a heterodimer of two "crossed" scFv fragments, in which the VH and VL domains of the two antibodies are located on different polypeptide chains. Biantibodies are described more fully in, for example: EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0101] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody), where residues from the hypervariable region (HVR) of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates, possessing the desired antibody specificity, affinity, and function. In some cases, frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor or donor antibody. These modifications are intended to further enhance antibody performance. Generally, the humanized antibody will contain substantially all, and typically two, variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. The humanized antibody also optionally contains at least a portion of the immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0102] In some embodiments, the methods described herein include one or more depth filtration steps. A depth filtration step is a chromatography technique that includes processing via a depth filter. In some embodiments, the depth filter comprises a porous filter media capable of retaining portions of a sample, such as cellular components and debris, wherein filtration occurs within a depth, for example, a filter material. In some embodiments, the depth filter comprises synthetic materials, non-synthetic materials, or combinations thereof. In some embodiments, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a viscous resin, and an ash composition. In some embodiments, at least a portion of the substrate of the depth filter comprises surface modification. In some embodiments, the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification, and anionic surface modification. In some embodiments, the depth filter is selected from the group consisting of EMPHAZE™ depth filters (such as the EMPHAZE™ AEX depth filter), X0SP depth filter, PDD1 depth filter, ZETA PLUS™ 120ZA depth filter, and ZETA PLUS™ 120ZB depth filter.

[0103] In some embodiments, the depth filter comprises cellulose fibers, diatomaceous earth, and perlite. In some embodiments, the depth filter comprises two layers, each containing a cellulose filter matrix, wherein the cellulose filter matrix is ​​impregnated with one or more filter aids comprising diatomaceous earth or perlite, and wherein each layer further comprises a resin binder. In some embodiments, the depth filter is a PDD1 depth filter.

[0104] In some embodiments, the depth filter comprises silica (such as a silica filter aid) and polyacrylate fibers. In some embodiments, the depth filter comprises two layers of filter media, wherein the first layer comprises silica (such as a silica filter aid) and the second layer comprises polyacrylate fibers (such as polyacrylate fiber pulp). In some embodiments, the depth filter is a depth filter that comprises synthetic materials but does not contain diatomaceous earth and / or perlite. In some embodiments, the depth filter is an XOSP depth filter.

[0105] In some embodiments, the silica filter aid is a precipitated silica filter aid. In some embodiments, the filter aid is an aspect of a filter (such as a layer) that helps perform filtration functions. In some embodiments, the silica filter aid is a silica gel filter aid. In some embodiments, the silica filter aid has about 50% silanol ionized at pH 7. In some embodiments, the silica filter aid is a silica gel filter aid wherein about 50% of the silanol in the silica filter aid is ionized at pH 7. In some embodiments, the silica filter aid is precipitated from silica (such as SIPERNAT® (Evonik Industries AG)) or silica gel (such as Kieseigel 60 (Merck KGaA)). In some embodiments, the polyacrylic fiber is a nonwoven polyacrylic fiber pulp. In some embodiments, the polyacrylic fiber is an electrospun polyacrylic nanofiber. In some embodiments, the degree of fibrillation of the polyacrylic fiber is related to a Canadian Standard Freeness (CSF) of about 10 mL to about 800 mL. In some embodiments, the pore size of the depth filter is from about 0.05 µm to about 0.2 µm, such as about 0.1 µm. In some embodiments, the surface area of ​​the depth filter is about 0.1 m². 2 To approximately 1.5 m 2 Such as approximately 0.11 m 2 Approximately 0.55 m 2 or about 1.1 m 2In some embodiments, the depth filter does not contain diatomaceous earth and / or perlite. In some embodiments, the depth filter comprises two layers of filter media, wherein the first layer comprises a silica filter aid having approximately 50% silanols ionized at pH 7, and the second layer comprises a polyacrylic cellulose pulp having a degree of polyacrylic cellulose fibrillation related to approximately 10 mL to approximately 800 mL of Canadian Standard Freeness (CSF), and wherein the depth filter does not contain diatomaceous earth.

[0106] In some embodiments, the depth filter comprises a hydrogel Q (quaternary ammonium)-functionalized nonwoven material and a multi-zone microporous membrane. In some embodiments, the depth filter comprises four layers comprising a hydrogel Q-functionalized nonwoven material and a nine-zone microporous membrane. In some embodiments, the nonwoven material comprises polypropylene. In some embodiments, the depth filter is a depth filter comprising synthetic materials but excluding diatomaceous earth and / or perlite. In some embodiments, the depth filter is an EMPHAZE™ AEX depth filter.

[0107] In some embodiments, the depth filter includes multiple components or layers. In some embodiments, the depth filter includes multiple layers, the multiple layers including one or more layers comprising anion exchange (AEX) functional polymers. In some embodiments, the layer comprising an AEX functional polymer comprises quaternary ammonium (Q), such as Q functional hydrogels. In some embodiments, the layer comprising an AEX functional polymer comprises a quaternary ammonium (Q) functional polymer associated with a nonwoven fabric. In some embodiments, the layer comprising an AEX functional polymer comprises a quaternary ammonium (Q) functional hydrogel covalently grafted onto a fine-fiber polypropylene nonwoven scaffold. In some embodiments, the depth filter includes multiple layers, the multiple layers including a layer comprising a multi-zone membrane comprising a nine-zone membrane with a pore size of about 0.05 µm to about 0.3 µm (such as about 0.22 µm). In some embodiments, the depth filter does not contain diatomaceous earth.

[0108] In some embodiments, the methods described herein include one or more hydrophobic interaction chromatography (HIC) steps. An HIC step is a chromatography technique that includes treatment with a HIC medium, such as a HIC filter or HIC column. In some embodiments, the HIC medium contains a hydrophobic portion comprising, for example, methyl, ethyl, propyl, octyl, or phenyl groups. In some embodiments, a sample is applied to the HIC medium in a polar buffer. In some embodiments, peptides are eluted from the HIC medium using a stepwise elution with an aqueous buffer that gradually decreases the salt concentration, gradually increases the detergent concentration, and / or adjusts the pH.

[0109] In some embodiments, the methods described herein are capable of reducing the rate of enzymatic hydrolysis activity of compositions obtained from a purification platform. In some embodiments, the rate of enzymatic hydrolysis activity represents the activity rate of one or more hydrolytic enzymes (such as one or more different hydrolytic enzymes). In some embodiments, the rate of enzymatic hydrolysis activity is an alternative measure of the activity of one or more enzymes in the composition. In some embodiments, the rate of enzymatic hydrolysis activity is measured by using an alternative substrate. In some embodiments, the rate of enzymatic hydrolysis activity is assessed by measuring the hydrolysis products of one or more hydrolytic enzymes.

[0110] As used herein, the terms “comprising,” “having,” “containing,” and “including,” as well as other similar forms and their grammatical equivalents, are intended to be equivalent in meaning and are open-ended, meaning that one or more items following any of these terms do not imply an exhaustive list of those items or that the invention is limited to only the listed items. For example, an article of manufacture “comprising” components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that “comprising” and its similar forms and grammatical equivalents include the disclosure of embodiments that “consistently consist of” or “comprises of.”

[0111] Where a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range (based on one-tenth of the lower limit unit, unless the context explicitly specifies otherwise) and any other stated or intermediate values ​​within the range are included in this disclosure and are subject to any explicit exclusions within the range. If a specified range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0112] In this document, references to “about” values ​​or parameters include (and describe) variations relating to that value or parameter itself. For example, a reference to “about X” includes a description of “X”.

[0113] As used herein (including the appended claims), the singular forms “a / an,” “or,” and “the / said” include plural references unless the context clearly specifies otherwise.

[0114] Purification Platform

[0115] In some aspects of this disclosure, purification platforms are provided that include depth filtration and / or hydrophobic interaction chromatography (HIC) steps. In some embodiments, a purification platform refers to a workflow for purifying a target to any extent from a sample containing the target. In some embodiments, the process workflow of the purification platform is the sequence of steps involved in purifying the target from a sample containing the target.

[0116] For the purposes of example and explanation of the disclosure herein, Figure 1A illustrates a sequential workflow of a portion of an exemplary purification platform 100. As shown in Figure 1A, the purification platform 100 includes sequential steps, including but not limited to a capture step 105, a conditioning step 110, one or more purification steps 115 (such as one or more peptide purification steps), a virus filtration step 120, and an ultrafiltration / percolation (UFDF) step 125. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more depth filtration steps. In some embodiments, the exemplary purification platform shown in Figure 1A includes a depth filtration step performed after the conditioning step 110 and before one or more purification steps. In some embodiments, the exemplary purification platform shown in Figure 1A includes a depth filtration step performed before the capture step 105. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more HIC steps. In some embodiments, the HIC step is performed after the one or more purification steps, after the pH maintenance step of the virus filtration step, and / or after the virus filtration step. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more depth filtration steps and one or more HIC steps.

[0117] Those skilled in the art will readily understand that the purification platform described herein guides a workflow for purifying a target from a sample containing the target, the components (assemblies) for each step of the workflow used to perform the purification platform, and the components and reagents used therein. In some instances of this disclosure, the purification platform and its usage methods are described in a modular manner. Such disclosure is not intended to limit the scope of this application. This disclosure covers any combination and / or arrangement of purification platforms encompassed by the individual components (assemblies) and / or steps disclosed herein.

[0118] Deep filtering steps

[0119] In some aspects, this disclosure provides a purification platform that includes a deep filtration step. As described herein, the deep filtration step may be placed at any of one or more locations within the purification platform. In some embodiments, the purification platform described herein includes one or more deep filtration steps located at any stage of the process workflow, such as any one of 2, 3, 4, or 5 deep filtration steps. In some embodiments, the purification platform includes more than one deep filtration step that is not performed in a direct sequence, i.e., no intervention steps of the purification platform are performed between deep filtration steps. In some embodiments, the purification platform includes more than one identical deep filtration step. In some embodiments, the purification platform includes more than one different deep filtration step, for example, including the use of different depth filters.

[0120] In some embodiments, the purification platform includes more than one depth filtration step, a first depth filtration step occurring before a capture step including treatment by protein A chromatography, and a second depth filtration step occurring after the capture step and before the purification step.

[0121] In some embodiments, the depth filtration step includes treatment through a depth filter. In some embodiments, the depth filter is a depth filter containing synthetic materials. Depth filtration steps (including the steps involved in treatment through a depth filter) are known in the art. See, for example, Yigzaw et al., Biotechnol Prog, 22, 2006 and Liu et al., mAbs, 2, 2010, which are hereby incorporated by reference in their entirety. Those skilled in the art will understand, for example, the components (assemblies), conditions, and reagents involved in the depth filtration step.

[0122] In some embodiments, the methods described herein include one or more depth filtration steps, each depth filtration step including treatment through a depth filter, wherein the depth filter is selected based on the pH of the solution entering the depth filter. In some embodiments, when the solution entering the depth filter is from about 5 to about 6.5, a depth filter comprising silica and polyacrylate fibers, such as an XOSP depth filter, is selected. In some embodiments, when the solution entering the depth filter is about 6.5 or less, such as about 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, or 5.0 or less, a depth filter comprising silica and polyacrylate fibers, such as an XOSP depth filter, is selected. In some embodiments, when the solution entering the depth filter is any one of about 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5.0, a depth filter comprising silica and polyacrylic fibers, such as the XOSP depth filter, is selected. In some embodiments, when the solution entering the depth filter is from about 7 to about 8.5, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane, such as the EMPHAZE™ depth filter, is selected. In some embodiments, when the solution entering the depth filter is about 7 or greater, such as about 7.1 or greater, 7.2 or greater, 7.3 or greater, 7.4 or greater, 7.5 or greater, 7.6 or greater, 7.7 or greater, 7.8 or greater, 7.9 or greater, 8.0 or greater, 8.1 or greater, 8.2 or greater, 8.3 or greater, 8.4 or greater, or 8.5 or greater, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane, such as the EMPHAZE™ depth filter, is selected. In some embodiments, when the solution entering the depth filter is any one of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane, such as the EMPHAZE™ depth filter, is selected. In some embodiments of the method described herein, the method may further include selecting the depth filter based on the pH of the solution entering the depth filter.Those skilled in the art will readily understand that, in some cases, the solution entering the depth filter and its properties may be based on a target, such as a peptide (e.g., an antibody), purified using one or more purification platforms described herein. Therefore, in some embodiments, the characteristics of the target (such as pI) are used as the basis for selecting a depth filter for the purification platform described herein.

[0123] In some embodiments, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a viscous resin, synthetic particles, an ionicly charged resin, and an ash composition. In some embodiments, the depth filter comprises diatomaceous earth. In some embodiments, the depth filter comprises an anion exchange medium.

[0124] In some embodiments, at least a portion of the substrate of the depth filter includes surface modification. In some embodiments, the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification, and anionic surface modification.

[0125] In some embodiments, the depth filter is selected from the group consisting of EMPHAZE™ depth filters (such as EMPHAZE™ AEX depth filter), X0SP depth filter, PDD1 depth filter, ZETA PLUS™ 120ZA depth filter and ZETA PLUS™ 120ZB depth filter.

[0126] HIC steps

[0127] In some aspects, this disclosure provides a purification platform that includes a HIC step. As described herein, the HIC step may be placed at any of one or more locations within the purification platform. In some embodiments, the purification platform described herein includes one or more HIC steps located at any stage of the process workflow, such as any of 2, 3, 4, or 5 HIC steps. In some embodiments, the purification platform includes more than one HIC step that is not performed in a direct sequence, i.e., no intervention steps of the purification platform are performed between HIC steps. In some embodiments, the purification platform includes more than one identical HIC step. In some embodiments, the purification platform includes more than one different HIC step, for example, including the use of different HIC media.

[0128] In some embodiments, the HIC step includes treatment with a HIC medium, such as a HIC column or HIC membrane. HIC steps (including those involving treatment with a HIC medium) are known in the art. See, for example, Liuet al. mAbs, 2, 2010, which is hereby incorporated by reference. Those skilled in the art will understand, for example, the components (assemblies), conditions, and reagents involved in the HIC step.

[0129] In some embodiments, the HIC medium comprises a hydrophobic resin. In some embodiments, at least a portion of the substrate of the HIC medium includes surface modification. In some embodiments, the surface modification is phenyl or butyl surface modification.

[0130] In some embodiments, the HIC step is a flow-through HIC step. In some embodiments, the HIC step is a binding and elution HIC step.

[0131] In some embodiments, the purification platform includes: one or more depth filtration steps at any stage of the process workflow; and one or more HIC steps at any stage of the process workflow.

[0132] Capture Steps

[0133] In some embodiments, the purification platform includes a capture step. In some embodiments, the capture step includes processing by affinity chromatography.

[0134] The capture steps (including those involved in processing by, for example, affinity chromatography) are known in the art. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0135] In some embodiments, affinity chromatography is selected from the group consisting of protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, protein XL chromatography, FcXL chromatography, κ chromatography, and κXL chromatography. In some embodiments, the capture step includes processing by protein A chromatography. In some embodiments, the capture step includes processing by FcXL chromatography.

[0136] In some embodiments, protein A chromatography is based on silica. In some embodiments, protein A chromatography is based on agarose. In some embodiments, protein A chromatography is based on organic polymers.

[0137] In some embodiments, protein A chromatography is selected from the group consisting of Prose vA™, Prosep® vA Ultra, Protein A Sepharose® Rapid Flow, MabSelect™, MabSelect™ SuRe, Poros® A, and MabCapture™.

[0138] Adjustment steps

[0139] In some embodiments, the purification platform includes a conditioning step. In some embodiments, the conditioning step is performed after the capture step.

[0140] The adjustment steps (including the steps involved in the processing of the adjustment steps) are known in the art. See, for example, Liuet al. mAbs, 2, 2010, which is incorporated herein by reference.

[0141] In some embodiments, the conditioning step includes a virus inactivation step, such as a low pH holding step. In some embodiments, the low pH holding step is performed at a pH of about 2.5 to about 4. In some embodiments, the low pH holding step is configured for virus inactivation. In some embodiments, the low pH holding step is capable of inactivating endogenous / exogenous viruses.

[0142] One or more purification steps

[0143] In some embodiments, the purification platform includes one or more purification steps. In some embodiments, the one or more purification steps are performed after the capture and conditioning steps. In some embodiments, the one or more purification steps include a peptide purification step. In some embodiments, the one or more purification steps include more than one (such as any one of 2, 3, 4, or 5) peptide purification steps.

[0144] Peptide purification procedures (including the steps involved in peptide purification processes) are known in the art. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0145] In some embodiments, the peptide purification step includes treatment by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induced chromatography, ceramic hydroxyapatite chromatography, and multi-component chromatography.

[0146] In some embodiments, the peptide purification step includes treatment by chromatography selected from the group consisting of: diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), trimethylaminoethyl (TMAE), quaternary ammonium, quaternary aminoethyl (QAE), thiopropylaryl (SP), SP-Sepharose® (crosslinked, bead-like agarose) fast flow (FF), SP-Sepharose® XL, quaternary ammonium (Q) Sepharose® FF, mercaptoethylpyridine (MEP)-Hypercel™, CaptoMMC (multiplex chromatography), Capto Adhere, Poros® XS, and Poros® 50HS.

[0147] In some embodiments, the peptide purification step is a binding and elution peptide purification step. In some embodiments, the peptide purification step is a flow-through peptide purification step. In some embodiments, the peptide purification step is a weak partition chromatography peptide purification step. In some embodiments, the peptide purification step is an overload peptide purification step.

[0148] Virus filtration steps

[0149] In some embodiments, the purification platform includes a virus filtering step. In some embodiments, the virus filtering step is performed after one or more purification steps.

[0150] Virus filtering steps (including the steps involved in the processing of virus filtering steps) are known in the art. See, for example, Liu et al. mAbs, 2, 2010 and U.S. Application No. 20140309403, which are incorporated herein by reference.

[0151] In some embodiments, the virus filtration step includes treatment through a virus filter. In some embodiments, the virus filtration step includes a pH maintenance step. In some embodiments, treatment through the virus filter is performed after the pH maintenance step.

[0152] UFDF steps

[0153] In some embodiments, the purification platform includes a UFDF step. In some embodiments, the UFDF step is performed after the one or more purification steps and / or after the virus filtering step.

[0154] UFDF steps (including the steps involved in processing UFDF steps) are known in the art. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0155] In some embodiments, the UFDF step includes processing via ultrafiltration. In some embodiments, the UFDF step is performed in tangential flow filtration (TFF) mode. In some embodiments, the UFDF step includes processing via tangential flow filtration, such as high-performance tangential flow filtration.

[0156] As described above, the purification platform disclosed in this application may include any combination and arrangement of purification steps, including those described herein. For example, in some embodiments, the purification platform includes a capture step and a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0157] In some embodiments, the purification platform sequentially includes: a capture step; and a conditioning step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0158] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as a HIC step performed after the one or more purification steps.

[0159] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; and a virus filtration step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the deep filtration step is performed after the one or more purification steps and before the virus filtration step. In some embodiments, the deep filtration step is performed after the virus filtration step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected after the one or more purification steps and / or after or during the pH maintenance step of the virus filtration step, or such a subsequent step.

[0160] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; and a UFDF step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the deep filtration step is performed after the one or more purification steps and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected from those performed after the one or more purification steps and / or after the UFDF step.

[0161] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; a virus filtering step; and a UFDF step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the deep filtration step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the deep filtration step is performed after the virus filtering step and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected after the one or more purification steps and / or after a pH maintenance step of a virus filtering step, after or during a virus filtering step, such as after and / or after a UFDF step.

[0162] In some embodiments, the purification platform includes a capture step and a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0163] In some embodiments, the purification platform sequentially includes: a capture step; and a conditioning step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0164] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0165] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; and a virus filtering step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the HIC step is performed after the virus filtering step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a deep filtering step, such as a deep filtering step performed after the capture step.

[0166] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; and a UFDF step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0167] In some embodiments, the purification platform sequentially includes: a capture step; a conditioning step; one or more purification steps; a virus filtering step; and a UFDF step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the HIC step is performed after the virus filtering step and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a deep filtering step, such as a deep filtering step performed after the capture step.

[0168] Samples, components and compositions obtained from the purification platform

[0169] In some respects, the purification platform described herein can be used to purify the target to any extent from a sample containing the target.

[0170] In some embodiments, the sample is a host cell sample. In some embodiments, the sample is host cell culture medium (HCCF). In some embodiments, the sample comprises a portion of host cell culture medium. In some embodiments, the sample is derived from host cell culture medium. In some embodiments, the sample comprises host cells. In some embodiments, the sample comprises components of host cells, such as host cell fragments. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is an *E. coli* cell.

[0171] In some embodiments, the sample has been processed, such as undergoing a treatment step prior to subjecting the sample to the purification platform described herein. In some embodiments, the sample contains a surfactant. In some embodiments, the sample contains a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0172] In some embodiments, the sample contains a target. In some embodiments, the target contains a peptide. In some embodiments, the target is a peptide. In some embodiments, the target is a peptide complex. In some embodiments, the target is an antibody moiety. In some embodiments, the antibody moiety is a monoclonal antibody. In some embodiments, the antibody moiety is a humanized antibody. In some embodiments, the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody. In some embodiments, the antibody portion is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0173] In some embodiments, the sample comprises one or more host cell proteins. In some embodiments, the host cell protein is a hydrolase. In some embodiments, the hydrolase is a lipase, esterase, thioesterase, phospholipase, or ceramidinase. In some embodiments, the hydrolase is a multi-enzyme protein. In some embodiments, the multi-enzyme protein is a fatty acid synthase. In some embodiments, the fatty acid synthase comprises a thioesterase subunit.

[0174] In some cases, the purification platform described herein may include multiple purification steps. In some embodiments, the term "composition" is used herein to describe any input (other than the initial sample input of the purification platform), intermediate, or output of any stage of the purification platform. For example, in some embodiments, the use of the term "composition" is not limited to describing the final output of the purification platform.

[0175] In some embodiments, the composition comprises a surfactant. In some embodiments, the composition comprises a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0176] In some embodiments, the composition comprises a target. In some embodiments, the target comprises a peptide. In some embodiments, the target is a peptide. In some embodiments, the target is a peptide complex. In some embodiments, the target is an antibody moiety. In some embodiments, the antibody moiety is a monoclonal antibody. In some embodiments, the antibody moiety is a humanized antibody. In some embodiments, the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody. In some embodiments, the antibody portion is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0177] In some embodiments, the composition comprises one or more host cell proteins. In some embodiments, the host cell protein is a hydrolytic enzyme. In some embodiments, the hydrolytic enzyme is a lipase, esterase, thioesterase, phospholipase, or ceramide enzyme.

[0178] Additional steps

[0179] In some respects, this disclosure provides additional steps involved in or related to the purification platform described herein. The additional steps involved in or related to the purification platform, and the methods for performing these steps, are known. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference in its entirety.

[0180] In some embodiments, the purification platform further includes a sample processing step, such as a sample preparation step. In some embodiments, the purification platform further includes a clarification step, such as clarifying HCCF. In some embodiments, the purification platform further includes a host cell and host cell debris removal step, such as removing host cells and host cell debris from the sample and / or composition obtained from the purification platform. In some embodiments, the purification platform further includes a centrifugation step. In some embodiments, the purification platform further includes a sterile filtration step. In some embodiments, the purification platform further includes a tangential flow microfiltration step. In some embodiments, the purification platform further includes a flocculation / precipitation step.

[0181] Methods using a purification platform

[0182] In some aspects, this disclosure describes methods for using the purification platform described herein. In some embodiments, the method includes subjecting a sample containing a target to the purification platform described herein.

[0183] In some aspects, this document provides a method for reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform described herein, comprising one or more deep filtration steps and / or one or more HIC steps. The method comprises subjecting a sample to the purification platform, thereby reducing the rate of enzymatic hydrolysis activity of the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction in the rate of enzymatic hydrolysis activity of the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps is at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the rate of enzymatic hydrolysis activity is the rate of hydrolysis of polysorbate enzymes.

[0184] In some aspects, this document provides a method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform described herein, comprising one or more deep filtration steps and / or one or more HIC steps. The method includes subjecting a sample to the purification platform to reduce the level of the hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction in the level of one or more hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps is at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate.

[0185] In some aspects, this document provides a method for reducing the degradation of polysorbate esters in compositions obtained from a purification platform described herein, comprising one or more depth filtration steps and / or one or more HIC steps. The method includes subjecting a sample to the purification platform, thereby reducing the degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction in the degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps is at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0186] In some aspects, this document provides a method for extending the shelf life of compositions obtained from a purification platform described herein, comprising one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby extending the shelf life of the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative extension of the shelf life of the composition compared to purifying the sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps is at least about 1 week, such as at least about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, or more than 24 months. In some embodiments, the shelf life of the composition is at least about 1 week, such as at least about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 ​​months, or more than 48 months, compared to purifying the sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the shelf life of the composition is more than 6 months, more than 9 months, more than 12 months, more than 18 months, more than 24 months, more than 30 months, more than 36 months, more than 42 months, more than 48 months, or more than 48 months.

[0187] In some respects, this document provides a method for producing a composition of polysorbate with less degradation, obtained from a purification platform described herein comprising one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform to produce a composition of polysorbate with less degradation compared to purifying a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0188] In some respects, this document provides a method for reducing the aggregation of targets in compositions obtained from a purification platform described herein, which includes one or more deep filtration steps and / or one or more HIC steps. This method involves subjecting a sample to the purification platform, thereby reducing the aggregation of targets in the composition compared to purifying a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0189] As described herein, one or more properties of a composition obtained from a purification platform that subjectes a sample to one or more deep filtration steps and / or one or more HIC steps are compared to a sample purified using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. Those skilled in the art will understand that, in some cases, such comparisons must be performed under appropriate conditions that allow for meaningful comparisons. For example, when comparing a composition obtained from a purification platform that includes one or more deep filtration steps and / or one or more HIC steps to a sample purified using the same purification platform without one or more deep filtration steps and / or one or more HIC steps, the time factor, which may affect the readings of the enzyme hydrolysis activity rate, must be considered. Other relevant factors to consider when comparing a composition to a reference include experimental conditions, the assay used, temperature conditions, pH, timing, sample, one or more buffers, and one or more sample sources.

[0190] In some embodiments, the method includes subjecting a sample containing a target to a purification platform including a capture step; and a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0191] In some embodiments, the method includes subjecting a sample containing a target to a purification platform, the purification platform sequentially comprising: a capture step; and a conditioning step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed before the capture step. In some embodiments, the depth filtration step is performed after the capture step and before the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step. In some embodiments, the purification platform further comprises a second depth filtration step. In some embodiments, the purification platform further comprises a HIC step.

[0192] In some embodiments, the method includes subjecting a sample containing a target to a purification platform, the purification platform sequentially comprising: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further comprises a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further comprises a HIC step, such as a HIC step performed after the one or more purification steps.

[0193] In some embodiments, the method includes subjecting a sample containing a target to a purification platform that sequentially includes: a capture step; a conditioning step; one or more purification steps; and a virus filtration step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the deep filtration step is performed after the one or more purification steps and before the virus filtration step. In some embodiments, the deep filtration step is performed after the virus filtration step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected after the one or more purification steps and / or after, during, or after the pH maintenance step of the virus filtration step.

[0194] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; and a UFDF step, wherein the purification platform further includes a depth filtration step. In some embodiments, the depth filtration step is performed before the capture step. In some embodiments, the depth filtration step is performed after the capture step and before the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if more than one purification step exists, the depth filtration step is performed between or after any of the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and before the UFDF step. In some embodiments, the depth filtration step is performed after the UFDF step. In some embodiments, the purification platform further includes a second depth filtration step, such as a depth filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected from those performed after the one or more purification steps and / or after the UFDF step.

[0195] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; a virus filtering step; and a UFDF step, wherein the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if more than one purification step exists, the deep filtration step is performed between or after any of the one or more purification steps. In some embodiments, the deep filtration step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the deep filtration step is performed after the virus filtering step and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, such as one or more HIC steps selected after the one or more purification steps and / or during the pH maintenance step of the virus filtration step, after the virus filtration step, or such as after and / or after the UFDF step.

[0196] In some embodiments, the method includes subjecting a sample containing a target to a purification platform, the purification platform including a capture step; and a HIC step. In some embodiments, the HIC step is performed prior to the capture step. In some embodiments, the HIC step is performed after the capture step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0197] In some embodiments, the method includes subjecting a sample containing a target to a purification platform, the purification platform sequentially comprising: a capture step; and a conditioning step, wherein the purification platform further comprises a HIC step. In some embodiments, the HIC step is performed prior to the capture step. In some embodiments, the HIC step is performed after the capture step and prior to the conditioning step. In some embodiments, the HIC step is performed after the conditioning step. In some embodiments, the purification platform further comprises a second HIC step. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0198] In some embodiments, the method includes subjecting a sample containing a target to a purification platform that sequentially includes: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0199] In some embodiments, the method includes subjecting a sample containing a target to a purification platform that sequentially includes: a capture step; a conditioning step; one or more purification steps; and a virus filtering step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the HIC step is performed after the virus filtering step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a deep filtering step, such as a deep filtering step performed after the capture step.

[0200] In some embodiments, the method includes subjecting a sample containing a target to a purification platform that sequentially includes: a capture step; a conditioning step; one or more purification steps; and a UFDF step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a depth filtration step, such as a depth filtration step performed after the capture step.

[0201] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; a virus filtering step; and a UFDF step, wherein the purification platform further includes a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step and before the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between or after any of the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and before the virus filtering step. In some embodiments, the HIC step is performed after the virus filtering step and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after the one or more purification steps. In some embodiments, the purification platform further includes a deep filtering step, such as a deep filtering step performed after the capture step.

[0202] For the purposes of example and explanation of the disclosure herein, Figure 1B illustrates a sequential workflow of options available for aspects of an exemplary purification platform 200. As shown in Figure 1B, the purification platform includes: a protein A chromatography step 210; a further chromatography step selected from HIC 225, cation exchange chromatography 225, or multivariate chromatography 230; and one or more depth filtration steps selected from any one of an EMPHAZE™ depth filtration step 205 of HCCF prior to the protein A chromatography step, an XOSP depth filtration step 215 of the protein A conjugate, or an EMPHAZE™ depth filtration step 220 of the protein A conjugate.

[0203] According to Figure 1B, in some embodiments, the purification platform includes an EMPHAZE™ deep filtration step 205 of HCCF prior to subjecting the EMPHAZE™ deep-filtered consumable to protein A chromatography 210. In such embodiments, the protein A consumable is subjected to an XOSP deep filtration step 215 or an EMPHAZE™ deep filtration step 220 prior to downstream chromatography steps. In some embodiments, the purification platform includes an XOSP deep filtration step 215, prior to which the protein A consumable is conditioned by adjusting the pH of the protein A consumable to approximately 5 to approximately 6.5. In some embodiments, the purification platform includes an XOSP deep filtration step 215, the XOSP deep-filtered consumable further subjected to HIC (such as phenyl SEPHAROSE® fast flow) or cation exchange chromatography (such as POROS® 50HS). In some embodiments, the purification platform includes an EMPHAZE™ deep filtration step 220, prior to which the protein A conjugate is conditioned by adjusting the pH of the protein A conjugate to approximately 7 to approximately 8.5. In some embodiments, the purification platform includes an EMPHAZE™ deep filtration step 220, and the EMPHAZE™ deep-filtered conjugate is further subjected to multivariate chromatography (such as Capto Adhere).

[0204] According to Figure 1B, in some embodiments, the purification platform does not include an EMPHAZE™ deep filtration step 205 for HCCF. In such embodiments, HCCF undergoes protein A chromatography 210, and the protein A consumable undergoes either an XOSP deep filtration step 215 or an EMPHAZE™ deep filtration step 220 prior to downstream chromatography steps. In some embodiments, the purification platform includes an XOSP deep filtration step 215, prior to which the protein A consumable is conditioned by adjusting the pH of the protein A consumable to approximately 5 to approximately 6.5. In some embodiments, the purification platform includes an XOSP deep filtration step 215, and the XOSP deep-filtered consumable is further subjected to HIC (such as phenyl SEPHAROSE® fast flow) or cation exchange chromatography (such as POROS® 50HS). In some embodiments, the purification platform includes an EMPHAZE™ deep filtration step 220, prior to which the protein A conjugate is conditioned by adjusting the pH of the protein A conjugate to approximately 7 to approximately 8.5. In some embodiments, the purification platform includes an EMPHAZE™ deep filtration step 220, and the EMPHAZE™ deep-filtered conjugate is further subjected to multivariate chromatography (such as Capto Adhere).

[0205] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a depth filtration step, comprising treatment with a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; (b) a capture step, comprising treatment with protein A chromatography; and (c) a purification step, wherein the purification step comprises treatment with chromatography selected from the group consisting of HIC, cation exchange chromatography, and multivariate chromatography. In some embodiments, the depth filter is an EMPHAZE™ depth filter. In some embodiments, the HIC is a phenyl SEPHAROSE® fast flow chromatography. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the multivariate chromatography is Capto Adhere.

[0206] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a first depth filtration step, comprising treatment through a first depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; (b) a capture step, comprising treatment through protein A chromatography; (c) a second depth filtration step, comprising treatment through a second depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; and (d) a purification step, comprising treatment through multivariate chromatography. In some embodiments, the first and second depth filters are EMPHAZE™ depth filters. In some embodiments, the multivariate chromatography is CaptoAdhere. In some embodiments, the second depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane is selected when the solution entering the depth filter is from about 7 to about 8.5.

[0207] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a capture step, including treatment by protein A chromatography; (b) a depth filtration step, including treatment by a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; and (c) a purification step, including treatment by multivariate chromatography. In some embodiments, the depth filter is an EMPHAZE™ depth filter. In some embodiments, the multivariate chromatography is Capto Adhere. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane is selected when the solution entering the depth filter is from about 7 to about 8.5.

[0208] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a first depth filtration step, including treatment through a first depth filter; (b) a capture step, including treatment through protein A chromatography; and (c) a second depth filtration step, including treatment through a second depth filter. In some embodiments, the first depth filter comprises a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane. In some embodiments, the first depth filter is an EMPHAZE™ depth filter. In some embodiments, the first depth filter comprises an inorganic filter aid, a cellulose and resin system. In some embodiments, the first depth filter is a 120ZB depth filter. In some embodiments, the second depth filter comprises silica and polyacrylic fibers. In some embodiments, the second depth filter is an XOSP depth filter. In some embodiments, the method further includes a conditioning step. In some embodiments, the method further includes one or more purification steps.

[0209] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a first depth filtration step, comprising treatment through a first depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; (b) a capture step, comprising treatment through protein A chromatography; (c) a second depth filtration step, comprising treatment through a second depth filter comprising silica and polyacrylate fibers; and (d) a purification step, comprising treatment through cation exchange chromatography. In some embodiments, the first depth filter is an EMPHAZE™ depth filter. In some embodiments, the second depth filter is an XOSP depth filter. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the second depth filter comprising silica and polyacrylate fibers is selected when the solution entering the depth filter is from about 5 to about 6.5.

[0210] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a first depth filtration step, comprising treatment through a first depth filter comprising a hydrogel Q-functionalized nonwoven medium and a multi-zone microporous membrane; (b) a capture step, comprising treatment through protein A chromatography; (c) a second depth filtration step, comprising treatment through a second depth filter comprising silica and polyacrylate fibers; and (d) a purification step, comprising treatment through HIC. In some embodiments, the first depth filter is an EMPHAZE™ depth filter. In some embodiments, the second depth filter is an XOSP depth filter. In some embodiments, the HIC is a phenyl SEPHAROSE® rapid flow chromatography. In some embodiments, the second depth filter comprising silica and polyacrylate fibers is selected when the solution entering the depth filter is about 5 to about 6.5.

[0211] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a capture step, including treatment by protein A chromatography; and (b) a depth filtration step, including treatment by a depth filter. In some embodiments, the depth filter comprises silica and polyacrylate fibers. In some embodiments, the depth filter is an XOSP depth filter. In some embodiments, the method further includes a conditioning step. In some embodiments, the method further includes one or more purification steps.

[0212] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising: (a) a capture step, including treatment by protein A chromatography; (b) a depth filtration step, including treatment by a depth filter comprising silica and polyacrylate fibers; and (c) a purification step, including treatment by cation exchange chromatography. In some embodiments, the depth filter is an XOSP depth filter. In some embodiments, the cation exchange chromatography is a POROS® 50HS. In some embodiments, a depth filter comprising silica and polyacrylate fibers is selected when the solution entering the depth filter is from about 5 to about 6.5.

[0213] In some embodiments, the method includes subjecting a sample containing the target to a purification platform comprising: (a) a capture step, including treatment by protein A chromatography; (b) a depth filtration step, including treatment by a depth filter comprising silica and polyacrylate fibers; and (c) a purification step, including treatment by HIC. In some embodiments, the depth filter is an XOSP depth filter. In some embodiments, the HIC is a phenyl SEPHAROSE® rapid flow chromatography. In some embodiments, a depth filter comprising silica and polyacrylate fibers is selected when the solution entering the depth filter is from about 5 to about 6.5.

[0214] Additional method steps

[0215] In some embodiments, the methods described herein further include additional method steps. In some embodiments, the method further includes a cell culture step. In some embodiments, the method further includes a formulation step, such as processing the composition to form a pharmaceutical composition or a precursor thereof.

[0216] In some embodiments, the method further includes determining the rate of enzymatic hydrolysis activity of the composition. In some embodiments, the method further includes performing a lipase activity assay on the composition obtained from the purification platform described herein. In some embodiments, the lipase activity assay includes measuring the lipase activity of one or more hydrolases by monitoring the conversion of a substrate, such as a non-fluorescent substrate, to a detectable product of the hydrolase, such as a fluorescent product. In some embodiments, the substrate comprises an ester bond. In some embodiments, the method further includes determining the product of one or more hydrolases, for example, as described in WO2018035025, which is hereby incorporated by reference in its entirety. In some embodiments, the method further includes determining the level of free fatty acids (FFA) in the composition obtained from the purification platform described herein by performing fatty acid mass spectrometry (FAMS). In some embodiments, the method further includes determining the level of one or more hydrolases in the composition. In some embodiments, the method further includes determining the shelf life of the composition. In some embodiments, the method further includes determining the level of target aggregates in the composition.

[0217] Pharmaceutical Composition

[0218] In some aspects, this disclosure provides pharmaceutical compositions obtained from the purification platforms described herein. In some embodiments, the pharmaceutical composition is obtained from the methods described herein. In some embodiments, the pharmaceutical composition is a purified composition. In some embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0219] In some embodiments, the pharmaceutical composition comprises an antibody moiety. In some embodiments, the pharmaceutical composition comprises an antibody moiety and a polysorbate. In some embodiments, the pharmaceutical composition comprises an antibody moiety, a polysorbate, and host cell impurities, such as host cell proteins, for example, hydrolases.

[0220] In some embodiments, the pharmaceutical composition comprises polysorbate. In some embodiments, the pharmaceutical composition is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0221] In some embodiments, the pharmaceutical composition has a reduced rate of enzymatic hydrolysis activity compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0222] In some embodiments, the pharmaceutical composition has reduced levels of one or more hydrolytic enzymes compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0223] In some embodiments, the pharmaceutical composition has reduced polysorbate degradation compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0224] In some embodiments, the pharmaceutical composition has an extended shelf life compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0225] In some embodiments, the pharmaceutical composition has less degraded polysorbate compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0226] In some embodiments, the pharmaceutical composition exhibits reduced target aggregation compared to a composition obtained by purifying the same sample using the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0227] The prepared antibody component composition

[0228] In some aspects, this disclosure provides formulated antibody fractional compositions obtained from the purification platforms described herein. In some embodiments, the formulated antibody fractional compositions are obtained from the methods described herein.

[0229] In some embodiments, the formulated antibody portion composition comprises an antibody portion. In some embodiments, the formulated antibody portion composition comprises an antibody portion and polysorbate. In some embodiments, the formulated antibody portion composition comprises an antibody portion, polysorbate, and host cell impurities, such as host cell proteins, for example, hydrolases.

[0230] In some embodiments, the antibody moiety compositions described herein have an extended shelf life compared to reference compositions, such as antibody moiety compositions formulated from the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the shelf life is assessed, such as by measuring, the aggregation of the antibody moiety of the formulated antibody moiety composition. In some embodiments, the shelf life is assessed, such as by measuring, the preservation of one or more functionalities of the antibody moiety of the formulated antibody moiety composition. In some embodiments, the shelf life is assessed, such as by measuring, the activity (e.g., binding activity) of the antibody moiety of the formulated antibody moiety composition.

[0231] In some embodiments, the formulated antibody moiety composition comprising an antibody moiety and polysorbate has a reduced polysorbate hydrolysis activity rate, wherein the shelf life of the composition exceeds about 12 months, such as exceeding any one of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. In some embodiments, the formulated antibody moiety composition has a reduced polysorbate hydrolysis activity rate compared to a reference, such as a formulated antibody moiety composition obtained from the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the reduced polysorbate hydrolysis activity rate is the reduced relative polysorbate hydrolysis activity rate.

[0232] In some embodiments, a formulated antibody moiety composition comprising an antibody portion and polysorbate has a reduced polysorbate hydrolysis activity rate, wherein the shelf life of the composition is extended compared to the shelf life specified in a document submitted to a health authority relating to the formulated antibody moiety composition, and wherein the extended shelf life is at least about 2 months, such as at least about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, compared to the shelf life specified in the document. In some embodiments, a formulated antibody moiety composition has a reduced polysorbate hydrolysis activity rate compared to a reference, such as a formulated antibody moiety composition obtained from the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the reduced polysorbate hydrolysis activity rate is a reduced relative polysorbate hydrolysis activity rate.

[0233] In some embodiments, a formulated antibody portion composition comprising an antibody portion and polysorbate has reduced polysorbate degradation compared to the degradation specified in documents submitted to health authorities relating to the formulated antibody portion composition, wherein the degradation is reduced by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, a formulated antibody portion composition has reduced polysorbate degradation compared to a reference, such as a formulated antibody portion composition obtained from the same purification platform without one or more depth filtration steps and / or one or more HIC steps. In some embodiments, the reduced polysorbate degradation is a reduced relative degradation of polysorbate.

[0234] In some embodiments, the polysorbate hydrolysis activity rate of the formulated antibody fraction composition is reduced by at least about 5% compared to the reference, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0235] In some embodiments, the formulated antibody portion composition comprises an antibody portion and polysorbate, wherein the polysorbate degrades by about 50% or less per year during storage of the liquid composition, such as about 45% or less per year, 40% or less per year, 35% or less per year, 30% or less per year, 25% or less per year, 20% or less per year, 15% or less per year, 10% or less per year, or 5% or less per year.

[0236] In some embodiments, compared to a reference antibody moiety such as an antibody moiety composition formulated from the same purification platform without one or more depth filtration steps and / or one or more HIC steps, the shortened aggregates of the antibody moiety compositions described herein form for at least about 6 months, such as at least about 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, at least about 20% less (such as at least about 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, or 100% less) of the antibody fractional composition formulated herein forms aggregates for at least about 6 months, such as at least about 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months, compared to a reference, wherein the reference is derived from a composition without one or more depth filtration steps and / or one or more HICs. The antibody fraction composition was prepared using the same purification platform as the steps. Methods for evaluation, such as measuring aggregate formation, are known in the art and include, for example, visual inspection, dynamic light scattering, static light scattering, and optical density measurement.

[0237] In some embodiments, the antibody fraction compositions formulated herein retain at least about 50% (such as at least about 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) of antibody fraction activity for at least about 6 months, such as at least about 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months, compared to a reference, wherein the reference is an antibody fraction composition formulated from the same purification platform without one or more depth filtration steps and / or one or more HIC steps.

[0238] In some embodiments, the antibody portion is a monoclonal antibody.

[0239] In some embodiments, the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0240] In some embodiments, the antibody is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0241] In some embodiments, the antibody portion is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0242] In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0243] Other aspects reported herein are formulated antibody compositions that exhibit oligosorbate degradation during storage. One aspect of the invention is a formulated antibody composition comprising an antibody / protein and a polysorbate, wherein the polysorbate degrades by 20% or less per year during the storage / shelf life of the formulated antibody composition (15% or less in one example, 12% or less in another, 10% or less in another, 9% or less in another, 8% or less in another, 7% or less in another, 6% or less in another, 5% or less in another, 4% or less in another, 3% or less in another, 2% or less in another, and 1% or less in another). In one embodiment, the polysorbate degrades by 10% or less per year during the storage of the liquid composition.

[0244] On the other hand, there is an antibody composition comprising an antibody and polysorbate, wherein after one year the polysorbate is present in the composition at a concentration of at least 80% (at least 85% in one embodiment, at least 88% in one embodiment, at least 90% in one embodiment, at least 91% in one embodiment, at least 92% in one embodiment, at least 93% in one embodiment, at least 94% in one embodiment, at least 95% in one embodiment, at least 96% in one embodiment, at least 97% in one embodiment, at least 98% in one embodiment, at least 99% in one embodiment) of the initial concentration, wherein the initial concentration is the concentration of the antibody when it is formulated or initially stored in the liquid composition.

[0245] Exemplary embodiments

[0246] Example 1. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step; and (b) a depth filtration step, thereby reducing the rate of enzymatic hydrolysis of the composition compared to purifying the sample using the same purification platform without the depth filtration step.

[0247] Example 2. The method according to Example 1, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

[0248] Example 3. The method according to Example 1 or 2, wherein the relative reduction in the rate of enzyme hydrolysis activity of the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0249] Example 4. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step; and (b) a deep filtration step, thereby reducing the level of hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the deep filtration step.

[0250] Example 5. The method according to Example 4, wherein one or more hydrolytic enzymes are capable of hydrolyzing polysorbate.

[0251] Example 6. The method according to Example 4 or 5, wherein the relative reduction in the level of one or more hydrolytic enzymes in the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0252] Example 7. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step; and (b) a deep filtration step, thereby reducing the degradation of polysorbate in the composition compared to purifying the sample using the same purification platform without the deep filtration step.

[0253] Example 8. The method according to Example 7, wherein the relative reduction in degradation of the polysorbate in the composition is at least about 5% compared to purifying the sample using the same purification platform without the depth filtration step.

[0254] Example 9. The method according to any one of Examples 1-8, wherein the purification platform is used to purify the target from the sample, wherein the sample contains the target and one or more host cell impurities.

[0255] Example 10. The method according to Example 9, wherein the target comprises a polypeptide.

[0256] Example 11. The method according to Example 9 or 10, wherein the host cell impurity is a host cell protein.

[0257] Example 12. The method according to any one of Examples 1-11, wherein the depth filtering step is performed before the capture step or the depth filtering step is performed after the capture step.

[0258] Example 13. The method according to any one of Examples 1-12, wherein the depth filtering step includes processing through a depth filter.

[0259] Example 14. The method according to Example 13, wherein the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a viscous resin, and an ash composition.

[0260] Example 15. The method according to Example 14, wherein at least a portion of the substrate of the depth filter includes surface modification.

[0261] Example 16. The method according to Example 15, wherein the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification and anionic surface modification.

[0262] Example 17. The method according to any one of Examples 14-16, wherein the depth filter is selected from the group consisting of EMPHAZE™ depth filter, PDD1 depth filter, ZETA PLUS™ 120ZA depth filter and ZETAPLUS™ 120ZB depth filter.

[0263] Example 18. The method according to any one of Examples 1-17, wherein the capture step includes processing by affinity chromatography.

[0264] Example 19. The method according to Example 18, wherein the affinity chromatography is selected from the group consisting of protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, FcXL chromatography, protein XL chromatography, κ chromatography and κXL chromatography.

[0265] Example 20. The method according to any one of Examples 1-19, wherein the purification platform further includes a virus inactivation step, wherein the virus inactivation step is performed after the capture step.

[0266] Example 21. The method according to Example 20, wherein the deep filtering step is performed after the virus inactivation step.

[0267] Example 22. The method according to any one of Examples 1-21, wherein the purification platform further includes another depth filtration step performed prior to the capture step.

[0268] Example 23. The method according to any one of Examples 1-22, wherein the purification platform further comprises one or more purification steps, and wherein the one or more purification steps are performed after the capture step, the deep filtration step and, if present, the virus inactivation step.

[0269] Example 24. The method according to Example 23, wherein the one or more purification steps include a peptide purification step.

[0270] Example 25. The method according to Example 23 or 24, wherein the purification platform further includes another depth filtration step performed before, between or after the one or more purification steps.

[0271] Example 26. The method according to any one of Examples 1-25, wherein the purification platform further includes an ultrafiltration / percolation (UFDF) step, and wherein the UFDF step is performed after the one or more purification steps.

[0272] Example 27. The method according to Example 26, wherein the purification platform further includes another depth filtration step performed before or after the UFDF step.

[0273] Example 28. The method according to any one of Examples 1-27, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step.

[0274] Example 29. The method according to Example 28, wherein the HIC purification step is performed before, between, or after one or more purification steps, if present.

[0275] Example 30. The method according to Example 28, wherein the HIC purification step is performed after the one or more purification steps and before the UFDF step, if present.

[0276] Example 31. The method according to Example 26 or 27, wherein the purification platform further includes a pH maintenance step, wherein the pH maintenance step is performed after the one or more purification steps, if present, and before the UFDF step.

[0277] Example 32. The method according to Example 31, wherein the purification platform further includes a virus filtration step, wherein the virus filtration step is performed after the pH maintenance step and before the UFDF step.

[0278] Example 33. The method according to Example 32, wherein the virus filtering step includes processing through a virus filter.

[0279] Example 34. The method according to Example 28, wherein the HIC purification step includes processing through a HIC filter.

[0280] Example 35. The method according to any one of Examples 23-34, wherein the one or more purification steps each independently comprise treatment by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induced chromatography, ceramic hydroxyapatite chromatography, and multi-component chromatography.

[0281] Example 36. The method according to any one of Examples 23-35, wherein the one or more purification steps each independently comprise treatment by chromatography selected from the group consisting of: DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel™, Capto MMC, and Capto Adhere.

[0282] Example 37. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in sequence: (a) a capture step comprising treatment by affinity chromatography; (b) a virus inactivation step; (c) a second peptide purification step; (d) a third peptide purification step; and (e) an ultrafiltration / percolation (UFDF) step, wherein the purification platform further comprises a deep filtration step performed at one or more of the following times: (i) before the capture step; (ii) after the capture step and before the virus inactivation step; (iii) after the virus inactivation step and before the second peptide purification step; (iv) after the second peptide purification step and before the third peptide purification step; or (v) after the third peptide purification step and before the ultrafiltration / percolation (UFDF) step; thereby reducing the rate of enzymatic hydrolysis of the composition compared to purifying the sample using the same purification platform without the deep filtration step.

[0283] Example 38. The method according to Example 37, wherein the purification platform further includes, in the following order, a pH maintenance step and a virus filtration step performed after the third peptide purification step and before the UFDF step.

[0284] Example 39. The method according to Example 38, wherein the virus filtering step includes processing through a virus filter.

[0285] Example 40. The method according to any one of Examples 37-39, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step performed at one or more of the following: (i) after the third peptide purification step and before the pH holding step; (ii) after the pH holding step and before the virus filtration step; or (iii) after the virus filtration step and before the UFDF step.

[0286] Example 41. The method according to any one of Examples 1-40, further comprising determining the rate of enzyme hydrolysis activity of the composition.

[0287] Example 42. The method according to any one of Examples 1-41 further includes determining the level of one or more hydrolytic enzymes in the composition.

[0288] Example 43. The method according to any one of Examples 1-42, wherein the composition comprises polysorbate.

[0289] Example 44. The method according to Example 43, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0290] Example 45. The method according to any one of Examples 1-44 further includes a sample processing step.

[0291] Example 46. The method according to any one of Examples 1-45, wherein the sample is or is derived from a cell culture sample.

[0292] Example 47. The method according to Example 46, wherein the cell culture sample comprises host cells, and wherein the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli cells.

[0293] Example 48. The method according to any one of Examples 1-47, wherein the sample comprises a host cell or a component derived from the host cell.

[0294] Example 49. The method according to any one of Examples 1-48, wherein the sample comprises one or more host cell proteins, and wherein one or more host cell proteins is a hydrolytic enzyme.

[0295] Example 50. The method according to Example 49, wherein the hydrolytic enzyme is a lipase, esterase, thioesterase, phospholipase or ceramide enzyme.

[0296] Example 51. The method according to any one of Examples 1-50, wherein the sample contains a target, and wherein the target is an antibody moiety.

[0297] Example 52. The method according to Example 51, wherein the antibody portion is a monoclonal antibody.

[0298] Example 53. The method according to Example 51 or 52, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0299] Example 54. The method according to any one of Examples 51-53, wherein the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0300] Example 55. The method according to any one of Examples 51-54, wherein the antibody portion is selected from the group consisting of: olizumab, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, olizumab, lanperizumab, lejinzumab, omalizumab, ranituzumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0301] Example 56. A pharmaceutical composition obtained by the method according to any one of Examples 1-55.

[0302] Example 57. A formulated antibody fraction composition comprising an antibody fraction and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, and wherein the composition has a shelf life exceeding 24 months.

[0303] Example 58. A formulated antibody fraction composition comprising an antibody fraction and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, wherein the shelf life of the composition is extended compared to the shelf life specified in a document submitted to a health authority relating to the formulated antibody fraction composition, wherein the shelf life is extended by at least 6 months compared to the shelf life specified in the document.

[0304] Example 59. A formulated antibody portion composition comprising an antibody portion, wherein the formulated antibody portion composition has reduced polysorbate degradation, wherein the degradation is reduced by at least about 20% compared to the degradation specified in documents submitted to health authorities relating to the formulated antibody portion composition.

[0305] Example 60. A formulated antibody portion composition comprising an antibody portion and a polysorbate, wherein the polysorbate degrades by 20% or less per year during storage of the liquid composition.

[0306] Example 61. An antibody fraction composition formulated according to any one of Examples 57-60, wherein the antibody fraction is a monoclonal antibody.

[0307] Example 62. An antibody fraction composition formulated according to any one of Examples 57-61, wherein the antibody fraction is a human antibody, a humanized antibody, or a chimeric antibody.

[0308] Example 63. An antibody fractional composition formulated according to any one of Examples 57-62, wherein the antibody is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0309] Example 64. An antibody fraction composition formulated according to any one of Examples 57-63, wherein the antibody fraction is selected from the group consisting of: ozoglucomancil, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, ozoglucomancil, lanperizumab, leginizumab, omalizumab, ranituzumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0310] Example 65. An antibody fractional composition formulated according to any one of Examples 57-64, wherein the polysorbate hydrolysis activity rate is reduced by at least about 20%.

[0311] Example 66. An antibody fractional composition formulated according to any one of Examples 57-65, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0312] Example 67. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the rate of enzymatic hydrolysis of the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

[0313] Example 68. The method according to Example 67, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

[0314] Example 69. The method according to Example 67 or 68, wherein the relative reduction in the rate of enzyme hydrolysis activity of the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0315] Example 70. A method for reducing the level of one or more hydrolases in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multivariate chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step and before the purification step; or after the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the level of one or more hydrolases in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

[0316] Example 71. The method according to Example 70, wherein one or more hydrolytic enzymes are capable of hydrolyzing polysorbate.

[0317] Example 72. The method according to Example 70 or 71, wherein the relative reduction in the level of one or more hydrolytic enzymes in the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0318] Example 73. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) a capture step comprising treatment by affinity chromatography; and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step, wherein each depth filtration step comprises treatment by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic acid fibers; (ii) hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the degradation of polysorbate in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

[0319] Example 74. The method according to Example 73, wherein the relative reduction in degradation of the polysorbate in the composition is at least about 5% compared to purifying the sample using the same purification platform without the depth filtration step.

[0320] Example 75. The method according to any one of Examples 67-74, wherein the depth filter comprising the silica and the polyacrylic fiber comprises a silica filter aid and a polyacrylic fiber pulp.

[0321] Example 76. The depth filter comprising the hydrogel Q-functionalized nonwoven medium and the multi-zone microporous membrane according to any one of Examples 67-74, comprising four layers, the four layers comprising the hydrogel Q-functionalized nonwoven material and the nine-zone microporous membrane.

[0322] Example 77. The method according to any one of Examples 67-74, wherein the depth filter comprising cellulose fibers, diatomaceous earth and perlite comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is ​​impregnated with a filter aid comprising one or more of diatomaceous earth or perlite, and wherein each layer further comprises a resin binder.

[0323] Example 78. The method according to any one of Examples 67-77, wherein the depth filter is selected based on the pH of the solution entering the depth filter.

[0324] Example 79. The method according to Example 78, wherein when the solution entering the depth filter is about 5 to about 6.5, the depth filter comprising the silica and the polyacrylic fiber is selected.

[0325] Example 80. The method according to Example 78, wherein when the solution entering the depth filter is about 7 to about 8.5, the depth filter comprising the hydrogel Q-functionalized nonwoven medium and the multi-zone microporous membrane is selected.

[0326] Example 81. The method according to any one of Examples 67-80 further includes selecting the depth filter based on the pH of the solution entering the depth filter.

[0327] Example 82. The method according to any one of Examples 67-81, wherein the purification platform sequentially comprises: a depth filtration step, the depth filtration step comprising treatment through the depth filter comprising the hydrogel Q-functionalized nonwoven medium and the multi-zone microporous membrane; a capture step, the capture step comprising treatment by protein A chromatography; and the purification step.

[0328] Example 83. The method according to Example 82, wherein the purification step includes treatment by the HIC.

[0329] Example 84. The method according to Example 83, wherein the HIC is phenyl SEPHAROSE® fast flow chromatography.

[0330] Example 85. The method according to Example 82, wherein the purification step includes treatment by the cation exchange chromatography.

[0331] Example 86. The method according to Example 85, wherein the cation exchange chromatography is POROS® 50HS.

[0332] Example 87. The method according to any one of Examples 67-86, wherein the purification platform further includes a second depth filtration step, the second depth filtration step comprising treatment through the depth filter comprising the silica and the polyacrylate fiber, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0333] Example 88. The method according to Example 82, wherein the purification step includes processing by the multivariate chromatography.

[0334] Example 89. The method according to Example 88, wherein the multivariate chromatography is Capto Adhere.

[0335] Example 90. The method according to Example 88 or 89, wherein the purification platform further includes a second depth filtration step, the second depth filtration step comprising treatment through the depth filter comprising the hydrogel Q-functionalized nonwoven medium and the multi-zone microporous membrane, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0336] Example 91. The method according to any one of Examples 67-90, wherein the purification platform is used to purify the target from the sample, wherein the sample contains the target and one or more host cell impurities.

[0337] Example 92. The method according to Example 91, wherein the target comprises a polypeptide.

[0338] Example 93. The method according to Example 91 or 92, wherein the host cell impurity is a host cell protein.

[0339] Example 94. The method according to any one of Examples 67-93, wherein the purification platform further includes a virus inactivation step, wherein the virus inactivation step is performed after the capture step.

[0340] Example 95. The method according to Example 94, wherein the one or more deep filtering steps are performed after the virus inactivation step.

[0341] Example 96. The method according to any one of Examples 67-95, wherein the purification platform further includes an ultrafiltration / percolation (UFDF) step, and wherein the UFDF step is performed after the purification step.

[0342] Example 97. The method according to any one of Examples 67-96 further includes determining the rate of enzyme hydrolysis activity of the composition.

[0343] Example 98. The method according to any one of Examples 67-97 further includes determining the level of one or more hydrolytic enzymes in the composition.

[0344] Example 99. The method according to any one of Examples 67-98, wherein the composition comprises polysorbate.

[0345] Example 100. The method according to Example 99, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

[0346] Example 101. The method according to any one of Examples 67-100 further includes a sample processing step.

[0347] Example 102. The method according to any one of Examples 67-101, wherein the sample is or is derived from a cell culture sample.

[0348] Example 103. The method according to Example 102, wherein the cell culture sample comprises host cells, and wherein the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli cells.

[0349] Example 104. The method according to any one of Examples 67-103, wherein the sample comprises a host cell or a component derived from the host cell.

[0350] Example 105. The method according to any one of Examples 67-104, wherein the sample comprises one or more host cell proteins, and wherein one or more host cell proteins is a hydrolytic enzyme.

[0351] Example 106. The method according to Example 105, wherein the hydrolytic enzyme is a lipase, esterase, thioesterase, phospholipase or ceramide enzyme.

[0352] Example 107. The method according to any one of Examples 67-106, wherein the sample contains a target, and wherein the target is an antibody moiety.

[0353] Example 108. The method according to Example 107, wherein the antibody portion is a monoclonal antibody.

[0354] Example 109. The method according to Example 107 or 108, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0355] Example 110. The method according to any one of Examples 107-109, wherein the antibody portion is selected from the group consisting of: anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0356] Example 111. The method according to any one of Examples 107-110, wherein the antibody portion is selected from the group consisting of: oligrin, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunitozumab, tirelumab, bevacizumab, rituximab, atezolizumab, oligrintozumab, lanperizumab, lejinzumab, omalizumab, ranituzumab, emecizumab, celuzumab, prasinzumab, RO6874281 and RO7122290.

[0357] Example 112. A pharmaceutical composition obtained by the method according to any one of Examples 67-111.

[0358] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. This disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of this disclosure to the specific procedures described herein.

[0359] Example

[0360] Example 1

[0361] This example demonstrates a comparison between two purification platforms for purifying the antibody trastuzumab from unregulated oncology using the following: (1) a standard purification platform; and (2) the same purification platform that includes an additional PDD1 depth filtration step after conditioning the eluent from affinity chromatography and before cation exchange (CEX) chromatography.

[0362] The standard purification platform (1) is performed twice and consists of the following sequential steps: affinity chromatography, eluent conditioning, cation exchange chromatography, anion exchange chromatography, and tangential flow filtration and conditioning of the resulting merged product from the anion exchange chromatography. The merged product names and descriptions for the purification process are shown in Table 1 below.

[0363] Table 1. Names and descriptions of purified merged products.

[0364]

[0365] The purification platform, which includes a PDD1 depth filtration step (2), was used twice. The hydrolytic activity of polysorbate at unregulated bulk levels was compared by free fatty acid mass spectrometry (FAMS), the methodology of which is disclosed in more detail in the Materials and Methods section.

[0366] For purification platforms that include a deep filtration step, the eluent is conditioned after affinity chromatography and before filtration through a PDD1 deep filter (Pall PDD1; SUPRAcap™-50 SC050PDD1 (lot number: 102992583); area: 22 cm²). The PDD1 deep filter is equilibrated using CEX equilibration buffer. Filtration of the conditioned affinity pool is pressure-controlled. The filtration step is performed at room temperature (15°C – 30°C). Trastuzumab is passed through the PDD1 filter. The PDD1 deep filter is rinsed with CEX equilibration buffer before and after use. The PDD1 deep filter is discarded after each use. The acceptable range for CEX equilibration buffer is: 0.020 - 0.040 M MES (2-(N-morpholino)ethanesulfonic acid), 0.042 - 0.048 M NaCl, pH 5.50 - 5.70, and conductivity 5.10 - 5.70 mS / cm. The operating conditions for the PDD1 depth filter are shown in Table 2 below.

[0367] Table 2. Operating conditions of PDD1 depth filter.

[0368]

[0369] Cation exchange chromatography (SP Sepharose® FF chromatography) was performed in binding and elution mode. The cation exchange step reduced the levels of antibody aggregates, antibody variants, CHO HCP impurities, DNA, leached protein A, and other process-related impurities. Antibody charge variants were washed from the column with an increasing sodium chloride concentration gradient, and trastuzumab was eluted using stepwise elution. All chromatographic steps were performed at ambient temperature (15°C – 30°C).

[0370] Before loading onto the cation exchange column, the affinity conjugate is conditioned to pH 5.5 ± 0.3 using a tris(hydroxymethyl)aminomethane (Tris) base. If the conjugate is over-titrated, it is conditioned to the specified pH with citric acid, and then the conductivity is adjusted to 3.5 ± 1.0 mS / cm by adding ultrapure water (if necessary). The cation exchange column is equilibrated with equilibration buffer and loaded with the conditioned affinity conjugate. After loading, the column is washed with equilibration buffer, followed by a gradient wash with increasing conductivity, and then washed again with equilibration buffer. Trastuzumab is eluted from the column by stepwise elution with elution buffer. Elution is started and stopped based on absorbance and volume.

[0371] The acceptable range for CEX equilibration buffer is: 0.020–0.040 M MES, 0.042–0.048 M NaCl, pH 5.50–5.70, and conductivity 5.10–5.70 mS / cm. The acceptable range for elution buffer is: 0.020–0.040 M MES, 0.092–0.098 M NaCl, pH 5.50–5.70, and conductivity 10.10–10.80 mS / cm. The operating conditions for cation exchange chromatography are shown in Table 3 below.

[0372] Table 3. Operating conditions for cation exchange chromatography.

[0373]

[0374] a Ktrastuzumab / SP Sepharose® cation exchange resin.

[0375] Anion exchange chromatography (Q Sepharose® chromatography) was performed in flow-through mode to reduce CHO HCP, DNA, protein A, and potential viruses. Trastuzumab was flow-through the column under the specified loading and washing conditions. All chromatographic steps were performed at ambient temperature (15°C – 30°C).

[0376] Adjust the pH of the cation exchange pool to pH 8.0 ± 0.5 using Tris base and MES (if needed), and adjust the conductivity to 5.5–7.8 mS / cm using ultrapure water. Equilibrate the anion exchange column with equilibration buffer, then load the pH-adjusted cation exchange pool. After loading, wash the column with equilibration buffer. Pooling is based on absorbance and volume. Adjust the pH of the anion exchange pool to 6.0 ± 0.1 using acetic acid.

[0377] The acceptable range for the equilibration buffer is: 0.015 - 0.035 M Tris, 0.025 - 0.075 M NaCl, and pH 7.5 - 8.5. The operating conditions for cation exchange chromatography are shown in Table 4 below.

[0378] Table 4. Operating conditions for anion exchange chromatography.

[0379]

[0380] The regulated anion exchange conjugate was subjected to tangential flow filtration (TFF) for concentration and percolation. To achieve a protein concentration of 30 ± 5 mg / mL from the unregulated bulk, the regulated anion exchange conjugate was concentrated using a TFF device equipped with a 30 kDa polyethersulfone (PES) membrane. Subsequently, the buffer composition was adjusted to meet the conditions by adding a solution containing histidine.

[0381] Before use, equilibrate the ultrafiltration membrane with percolation buffer. Concentrate the conditioned anion exchange pool to an intermediate concentration of 10–50 g / L and percolate in a TFF unit with at least 8 pool volumes of percolation buffer. Subsequently, adjust the buffer composition to 0.02 mol / L histidine / histidine HCl, pH 5.3 ± 0.2 by adding the appropriate amount of conditioning buffer. If necessary, adjust the protein concentration to 30 ± 5 mg / mL by adding percolation buffer.

[0382] The percolation buffer was 0.02 mol / L histidine / histidine-HCl, pH 5.3 ± 0.2. The operating conditions for cation exchange chromatography are shown in Table 5 below.

[0383] Table 5. TFF Operating Conditions.

[0384]

[0385] The amount of host cell protein in the cation exchange chromatography loading composition was measured, and the results are provided in Table 6. Both parallel assays using the purification platform with the PDD1 depth filter showed a decrease in host cell protein levels compared to a conventional purification platform.

[0386] Table 6. Host cell proteins measured in cation exchange chromatography loading compositions.

[0387]

[0388] The amount of host cell protein in the TFF pool after filtration conditioning was measured, and the results are presented in Table 7. Both parallel assays using the purification platform with the PDD1 depth filter showed reduced host cell protein levels compared to a conventional purification platform.

[0389] Table 7. Host cell proteins of TFF conjugates after conditioning with the percolation composition.

[0390]

[0391] Hydrolytic activity in the TFF confluent after percolation was measured, and the results are presented in Figure 2. Hydrolytic activity was measured using FAMS at 40 °C, 0.04% (w / v) SR-PS20, 10 mM methionine, 100 mM Tris, pH 8.0, and a final trastuzumab concentration of 6 g / L. Compared to parallel assays on a conventional purification platform, two parallel assays on the PDD1 deep filtration purification platform showed a decrease in the rate of enzyme hydrolytic activity, as indirectly measured by the amount of free fatty acids. Figure 2 ).

[0392] Materials and Methods

[0393] Protein concentration determination. Protein concentration was determined by UV spectroscopy using a Cary® 50 UV-Vis spectrophotometer (Varian) or NanoDrop™ OneC (Thermo Scientific). Protein samples were diluted in their respective buffers and measured twice. Concentration was determined according to the following equation derived from Lambert-Beer's law: = (280 nm – 320 nm) / ∙ ∙ F, where protein concentration [mg / ml], absorbance, ε extinction coefficient [ml / (mg·cm)], cell length [cm], and F dilution factor. The specific extinction coefficients for trastuzumab, faliximab, and FAP-IL2v were 1.48, 1.7, and 1.35 ml / (mg·cm), respectively.

[0394] Lipase activity assay (LEAP assay). Lipase activity was measured by monitoring the conversion of a non-fluorescent substrate (4-MU, Chem Impex Int'l Inc.) to a fluorescent product (MU, Sigma-Aldrich) via substrate ester bond cleavage. The protein conjugate sample to be analyzed was reburied to 150 mM Tris-Cl pH 8.0 using an Amicon Ultra-0.5 ml centrifuge filter (10,000 Da cutoff, Merck Millipore). The assay reaction mixture contained 80 µL of reaction buffer (150 mM Tris-Cl pH 8.0, 0.25% (w / v) Triton X-100, and 0.125% (w / v) gum arabic), 10 µL of 4-MU substrate (1 mM DMSO solution), and 10 µL of protein conjugate sample. The protein conjugate sample concentration was adjusted to 10–30 g / L and tested at three different concentrations. Each reaction was performed in triplicate in 96-well half-area polystyrene plates (black with caps and clear flat bottoms, Corning Incorporated), with the fluorescence signal increase monitored every 10 minutes by incubating the plates at 37°C for two hours in an Infinite 200Pro plate reader (Tecan Life Sciences). The MU generation rate was derived from the slope of the fluorescence time progression (0.5 h – 2 h) and represents the initial rate of the reaction (kJ / μL). raw [RFU / h]).

[0395] An enzyme blank reaction was also established to measure any non-enzymatic cleavage of the substrate induced by the buffer matrix. 10 µL of protein complex sample was replaced with 10 µL of 150 mM Tris-Cl at pH 8.0 in the reaction mixture. The autolytic cleavage rate (kJ / L) was then measured. self The lysis rate (RFU / h) was derived from the slope of the fluorescence time progression (0.5 h–2 h). To convert the fluorescence signal (RFU) to µM of MU, standard MU was added three times per plate. 10 µL of 150 mM Tris-Cl pH 8.0 and 80 µL of reaction buffer were added to 10 µL of MU (100 µM DMSO solution). The conversion factor a [RFU / µM] was calculated by averaging the fluorescence signal (0.5 h–2 h) and dividing it by the final concentration of MU present in the wells.

[0396] The lipase activity of a sample, given in [µM MU / h], is derived from the reaction rate (kJ / h) of the sample. raw The reaction rate (k) minus the enzyme blank in [RFU / h]) self -Cytosis [RFU / h]), and determined by dividing this by the conversion factor a [RFU / µM] to convert the fluorescence signal to µM MU / h. Activity was normalized to the protein concentration applied per well. To report hydrolytic activity as a percentage, the lipase activity of the reference sample was set to 100%.

[0397] Free fatty acids and FAMS determination. To monitor the content of free fatty acids after PS20 degradation in each elution fraction, samples were first prepared for PS20 stability studies and then analyzed by mass spectrometry. Unless otherwise specified, protein pool samples were adjusted to the same protein concentration (as shown in the experimental instructions) containing 0.04% (w / v) SR-PS20, 10 mM L-methionine, and 100 mM Tris at pH 8. L-methionine was added as an effective antioxidant to control the oxidative degradation of PS20 during the experimental time. As a buffer control, the applied protein volume was replaced with the same volume of the corresponding elution buffer system.

[0398] All reaction mixtures were incubated in a Thermomixer (Eppendorf) at 37°C or 40°C with shaking at 600 rpm. Samples were removed after the defined time points (as shown in the figures) and stored at -80°C until subsequent analysis.

[0399] Transfer 50 µL of sample to a new Eppendorf cup. Add 200 µL of FFA solvent solution (500 ng / mL D). 23 - Lauric acid and 500 ng / mL 13 C 14-Myristic acid in acetonitrile solution) and briefly vortex. Centrifuge the sample at 14,000 rpm for 5 min and then transfer to an HPLC vial for MS analysis. Fatty acids were separated from the injected sample using an ACQUITY UPLC® Peptide BEH C18 column (1.7 µm 2.1 x 150 mm and 300 Å) on a ThermoScientific™ Vanquish™ UHPLC system using 5 µL of the injected sample. Eluent A (0.1% ammonium hydroxide aqueous solution) and eluent B (100% acetonitrile) were used in the following gradient at a flow rate of 0.3 mL / min and a column temperature of 60 °C. The initial condition was 70% eluent B. The gradient was linearly changed from 0.2 min to 5.5 min, with eluent A increased to 100% and held to 6.0 min. Eluent B was set to 70% at 6.1 min and held to 10.0 min to reach equilibration. The mass spectrometer (Triple TOF® 6600, AB Sciex) operated in negative ionization mode with an ion spray voltage of –4500 V. The source temperature was set to 450 °C, and the TOF mass range was 100–1000 m / Z. The declustering potential was -120 V, and the collision energy was -10 V.

[0400] Large amounts of lauric acid, myristic acid, and isotopically labeled (D) were generated. 23 )-Lauric acid and ( 13 C 14 XIC of myristic acid. Integrate each peak and determine the relationship between lauric acid and D. 23 - Peak area ratio between lauric acid and myristic acid 13 C 14 - Peak area ratio between myristic acid and lauric acid. The peak area ratio is used to calculate the concentrations of lauric acid and myristic acid in the sample. Measurements are repeated twice. To report the amount of FFA (lauric acid (LA) and myristic acid (MA)) as a percentage, the amount of the reference sample is set to 100%.

[0401] Example 2

[0402] This example demonstrates a comparison between three platforms for purifying the antibody trastuzumab using the following: (1) a standard purification platform; (2) a standard purification platform with an added PDD1 deep filtration step performed after conditioning the eluent from affinity chromatography and before cation exchange (CEX) chromatography; and (3) a standard purification platform with an added EMPHAZE™ deep filtration step performed after conditioning the eluent from affinity chromatography and before cation exchange (CEX) chromatography.

[0403] A typical purification platform (1) consists of the following: affinity chromatography, eluent conditioning, cation exchange chromatography, anion exchange chromatography, and tangential flow filtration and conditioning of the combined product obtained from anion exchange chromatography.

[0404] For purification platforms that include a depth filtration step, the eluent is conditioned after affinity chromatography and before filtration through a depth filter. The PDD1 depth filter used was Pall PDD1, SUPRAcap™-50SC050PDD1 (lot number: 102992583), with an area of ​​22 cm². The EMPHAZE™ depth filter used was EMPHAZE™AEX Hybrid (lot number: S210650302), with an area of ​​25 cm². Trastuzumab flowed through the depth filter. The filtration step was performed at ambient temperature (15°C–30°C).

[0405] Before use, equilibrate the depth filter with cation exchange equilibration buffer. Filtration of the conditioned affinity conjugate is flow rate controlled. No rinsing is performed after filtration. This allows for examination of the actual reduction in host cell proteins (enzymes). Discard the filter after each use. The acceptable range for CEX equilibration buffer is: 0.020–0.040 M MES, 0.042–0.048 M NaCl, pH 5.50–5.70, and conductivity 5.10–5.70 mS / cm. Operating conditions for the depth filter are shown in Table 8 below.

[0406] Table 8. Depth Filter Operating Conditions.

[0407]

[0408] The amount of host cell protein in the cation exchange chromatography loading composition was measured, and the results are provided in Table 9. Protein concentration measurements were performed according to Example 1. Reduced host cell protein levels were observed on both the purification platform with the EMPHAZE™ depth filter and the purification platform with the PDD1 depth filter compared to the conventional purification platform.

[0409] Table 9. Host cell proteins measured in cation exchange chromatography loading compositions.

[0410]

[0411] Lipase activity was compared between cation exchange chromatography loading on a standard platform (affinity chromatography, no depth filtration, reference) and corresponding cation exchange chromatography loading including an additional depth filtration step after affinity chromatography (Figure 3). Lipase activity was determined according to Example 1.

[0412] The hydrolytic activity of cation exchange chromatography loading (after affinity chromatography, without depth filtration, reference) on a standard platform was compared with that of corresponding cation exchange chromatography loading including an additional depth filtration step after affinity chromatography (Figure 4). FAMS analysis was performed according to Example 1. Hydrolytic activity was measured using FAMS at 40 °C, 0.04% (w / v) SR-PS20, 10 mM methionine, 100 mM Tris, pH 8.0, and a final trastuzumab concentration of 4.8 g / L. Compared to the standard purification platform, both purification platforms, including the depth filter, showed a reduced rate of enzyme hydrolytic activity as measured by lipase activity assay (Figure 3) and a reduced amount of FAA in the FAMS assay (Figure 4).

[0413] Example 3

[0414] This example demonstrates the purification of anti-VEGF / Ang2 antibody using a purification platform employing a deep filtration step of HCCF (harvested cell fluid) prior to sterilization and a second deep filtration step following affinity chromatography (CaptureSelect™ FcXL). The purification platform used is detailed in Figure 5. A reference control was performed using the purification process shown, without the additional HCCF deep filtration step (Figure 5).

[0415] As shown in Figure 5, HCCF is filtered through three filters of different depths designed to remove potential host cell proteins. Antibodies flow through the filters. The filtration process is performed at ambient temperatures (15°C–30°C).

[0416] Before use, the three depth filters were equilibrated with affinity equilibration buffers of EMPHAZE™ (EMPHAZE™ AEX Hybrid (lot number: S228585702), area: 25 cm²), VR02 (BioCap_VR02 (lot number: 3923452), area: 25 cm²), and 120ZB (BioCap_120ZB (lot number: 3923452), area: 25 cm²) and cation exchange equilibration buffer of PDD1 (SUPRAcap™-50 SC050PDD1 (lot number: 103119429), area: 22 cm²). HCCF filtration was pressure-controlled (feed pressure = 0.2 MPa; maximum feed flow rate: 25 mL / min). Filtration of the regulated affinity conjugate was flow-controlled (feed flow rate = 5.2 ml / min; pressure control: 0.2 MPa). After filtration, rinse the filter with the same buffer to recover the product. Discard the filter after each use. Equilibration buffers are provided in Table 10.

[0417] Table 10. Depth filter equilibration buffer.

[0418]

[0419] Use 200 mL (~90 L / m) 2 Rinse the PDD1 filter with water for injection. Use 200 mL (~90 L / m³) 2 Rinse the EMPHAZE™ filter with C1 equilibration buffer. The filtered HCCF volume is 1750 mL (for EMPHAZE™, ~700 L / m²).

[0420] The operating conditions for CaptureSelect FcXL are shown in Table 11 below.

[0421] Table 11. Operation conditions for CaptureSelect FcXL.

[0422]

[0423] The amount of host cell protein loaded was measured at different points on the purification platform (see Figure 5), and the results are presented in Table 12.

[0424] Table 12. Measured host cell proteins in the compositions obtained on the purification platform. Asterisks indicate sampling points as shown in Figure 5.

[0425]

[0426] As described in Example 1, hydrolytic activity was measured using a lipase activity assay. Compared to a standard purification platform without an additional depth filter, the use of any test depth filter (EMPHAZE) significantly improved the performance. TM The enzymatic hydrolysis rate of FcXL eluates was decreased on different purification platforms (VR02 and 120ZB) (Figure 6A). Compared with a standard purification platform without an additional depth filter, the rate of enzymatic hydrolysis of FcXL eluates was decreased when using any of the test depth filters (EMPHAZE). TM The rate of enzymatic hydrolysis activity of PDD1 filtrate was reduced on different purification platforms (VR02 and 120ZB) (Figure 6B).

[0427] FAMS assays were performed to compare two purification platforms used for purifying the antibody from the strong cation exchange chromatography pool: (1) a standard purification platform; and (2) the same purification platform including an additional 120 ZB deep filtration step prior to affinity chromatography. The standard purification platform consisted of the following sequential steps: affinity chromatography, eluent conditioning, deep filtration, multi-component anion exchange chromatography, strong cation exchange chromatography, and tangential flow filtration. FAMS assays were performed according to Example 1 and under the following conditions: 37 °C, 0.04% (w / v) SR-PS20, 10 mM methionine, 150 mM Tris, pH 8.0, and a final antibody concentration of 50 g / L. Compared to the standard purification platform, the purification platform with the 120 ZB deep filter showed a reduced rate of enzymatic hydrolysis activity, as measured by the amount of free fatty acids (Figure 7).

[0428] Example 4

[0429] This example demonstrates a comparison of purification platforms used to purify anti-FAP-IL2v, which incorporate two different depth filters, X0SP or PDD1, for filtering regulated affinity chromatography (protein A chromatography) eluents.

[0430] Prepare and filter HCCF samples as described in Example 1.

[0431] The lipase activity of protein A chromatography eluents from a standard platform (without depth filtration) was compared with that from an affinity chromatography eluent from a purification platform including depth filtration, where the protein A chromatography eluents underwent either an XOSP or PDD1 depth filtration step. Lipase activity was determined according to Example 1. The lipase activity results from the fraction obtained from the XOSP depth filter are shown in Figure 8A. The lipase activity results from the PDD1 depth filter are shown in Figure 8B.

[0432] Example 5

[0433] This example demonstrates purification optimization experiments for purifying various antibody moieties, conducted to identify options that minimize polysorbate hydrolytic degradation in antibody moieties obtained from the purification platform. The experimental evaluations disclosed herein include depth filters such as EMPHAZE™ as protein A and second-stage chromatography column loading filters, and HIC media (SARTOBIND® phenyl membrane) as polishing column elution pooling filters or loading filters for subsequent virus filtration steps.

[0434] Several depth filters, including EMPHAZE™ and X0SP, were evaluated for their potential to remove or reduce hydrolases that lead to polysorbate degradation. The addition of the EMPHAZE™ filter was evaluated at two treatment levels within a standard mAb purification process. The first option was as a protein A loading filter prior to protein A chromatography, where HCCF was filtered before loading onto the protein A column. As summarized in Table 13, filtering HCCF prior to protein A chromatography resulted in a relative reduction of over 40% in hydrolytic activity compared to the standard purification process.

[0435] Table 13. Relative hydrolytic activity of compositions obtained from the purification platform.

[0436]

[0437] To demonstrate that the reduction in polysorbate degradation achieved using EMPHAZE™ was independent of the reduction in host cell protein alone, HCCF samples were purified via protein A at increased EMPHAZE™ throughput, and the CHOP and polysorbate degradation activity of the pooled samples were analyzed. As shown in Figure 9, the decrease in CHOP value depended on the EMPHAZE™ filtration throughput, while the significant decrease in the polysorbate degradation rate remained relatively constant. Although the CHOP value continued to increase with increasing EMPHAZE™ clarification throughput, reaching almost the same level as the control, the significant reduction in the achieved polysorbate degradation activity remained relatively stable compared to the control, reaching a maximum of 800 L / m³. 2 Flux.

[0438] The second option evaluated was to place the EMPHAZE™ and X0SP depth filter downstream of the virus inactivation step, or as a second... The loading filter for column chromatography. For this evaluation, the protein A confluence from multiple molecules is neutralized to pH 5.5 or pH 8.0 and filtered to 300 L / m³ using an EMPHAZE™ or XOSP filter. 2 Flux. The polysorbate hydrolytic activity of the filtered pooled consumables was compared with that of the unfiltered control. As summarized in Table 14, both the EMPHAZE™ and X0SP filters showed a significant reduction in polysorbate degradation compared to the unfiltered control pooled consumables. As shown in Figure 10, the polysorbate hydrolytic activity (measured using lipase activity assays during the anti-Tau mAb purification of the protein A pooled consumables) depended on the filtration flux of the X0SP depth filter at pH 5.5.

[0439] Table 14. Hydrolytic activity of compositions obtained from the purification platform.

[0440]

[0441] The use of HIC membranes has been evaluated as a method to reduce polysorbate degradation. HIC membranes can be placed after the final peptide chromatography column step (e.g., anion exchange chromatography), after a pH maintenance step (e.g., pH 5-6) before virus filtration, and / or after a virus filtration step before the UFDF step.

[0442] The primary HIC membrane evaluated in this study was the SARTOBIND® phenyl membrane. After adjustment to different pH values, the final peptide chromatographic pools were filtered through a SARTOBIND® membrane filter. The relative polysorbate degradation of the filtered pools was analyzed. As shown in Figures 11A to 11C, the relative polysorbate activity was significantly reduced compared to the control. The same data also indicated that the reduction in activity was dependent on the membrane volume flux (Figures 11A to 11C). Specifically, Figure 11A shows the specific activity of ozoglucomab at different fluxes for polysorbate degradation at pH 5.5. Specifically, Figure 11B shows the specific activity of celumab at different fluxes for polysorbate degradation at pH 5.5. Specifically, Figure 11B shows the specific activity of tocilizumab at different fluxes for polysorbate degradation at pH 6.5 (Figure 11C).

[0443] Materials and Methods

[0444] Resins Pro A Sepharose® FF, Fractogel® TMAE, and ceramic hydroxyapatite resin were purchased from GE Healthcare (Uppsala, Sweden), TOSOH Biosciences (King of Prussia, PA), and Bio-Rad (Hercules, California), respectively. Amicon centrifugal filters and XOSP depth filters were from Millipore (Bedford, MA). EMPHAZE™ AEX depth filters were from 3M (Meriden, CT), and SARTOBIND® phenyl membranes were from Sartorius (Bohemia, NY). 4-Methylumbelliferone octanoate, Triton™ X-100, and gum arabic were from Research Organcis (Cleveland, OH) and Acros Organics (Bridgewater, NJ), respectively. Ultra-refined (SR) grade PS20 for lipase activity assays was from Croda (Newark, NJ). All monoclonal antibodies reported here are humanized or human IgG1 expressed in CHO cells and produced by Roche (South San Francisco or Oceanside, CA).

[0445] For small EMPHAZE™ and other depth filters, use 25 cm. 2 Capsules of the desired size. The filter was first rinsed with 25 mM Tris and 250 mM NaCl at pH 7.5 at a flow rate of 8 ml / min at 100 L / m². After equilibration, the eluted mixture was filtered to 800 L / m² with HCCF (Pro A loaded) or neutralized Protein A. 2 per 100 L / m 2 Fractions were collected and purified by subsequent column steps, and polysorbate degradation activity was measured.

[0446] For evaluation of the SARTOBIND® phenyl membrane, a 3 ml size apparatus was used. The membrane was first washed with 30 ml of equilibration buffer at a flow rate of 15 L / min. After equilibration, the final chromatographic pool was filtered through the SARTOBIND® phenyl membrane, the fractions were collected, and the relative polysorbate degradation activity was determined.

[0447] All small-scale chromatography runs were performed on 0.66 cm x 20 cm columns. For protein A purification, the column was first pre-equilibrated in equilibration buffer, and then HCCF was loaded onto 10–20 g / L resin. After loading, the column was washed with >3 column volumes of equilibration buffer and >4 column volumes of wash buffer. Bound proteins were eluted with 2.5 mM HCl at pH 2.7 or 150 mM acetic acid. The elution fraction from 0.5 OD to 0.5 OD was collected, neutralized to pH 5, and polysorbate degradation activity was determined.

[0448] Lipase activity was assessed following the cleavage of ester bonds in an umbelliferone substrate with a polysorbate-like structure. In this study, a reaction mixture was prepared by mixing 10 µL of 10 mM 4-methylumbelliferone octanoate in DMSO with 80 µL of reaction buffer (50 mM Tris, pH 8.0, 0.4% Triton™ X-100, and 0.1% gum arabic). Fluorescence excitation and emission wavelengths were set at 355 nm and 460 nm, respectively. Fluorescence kinetics were continuously monitored at 37 °C for 2–4 h. Lipase activity for each sample was determined by calculating the initial reaction rate after kinetic solidification using linear fitting, corrected for the reaction rate against buffer only to account for background hydrolysis. Specific activity was determined by dividing the reaction rate by the sample protein concentration.

[0449] Protein samples were aseptically filtered using a 0.2 µm fluorodyne injection filter and spiked with 25x adjustment buffer (20 mg / mL methionine, 1% w / v SR PS20 in 10 mM histidine acetate solution, pH 5). The spiked samples were aliquoted into Eppendorf tubes under aseptic conditions and incubated at 25°C for up to 20 days. At each time point, aliquots were collected and frozen at -70°C until free fatty acid extraction was performed.

[0450] Free fatty acids in each sample were extracted with an acetonitrile solution containing an isotopically labeled internal standard for FFA. After centrifugation at 14,000 rpm for 5 min, the supernatant was transferred to an HPLC vial with a glass insert and frozen until measurement. A Waters H-class Bio UPLC system with a Waters ACQUITY UPLC® BEH300 C18 (1.7 μm, 2.1 x 150 mm) column was used in combination with an AB Sciex 6600 mass spectrometer for FFA detection. Free fatty acids (FAA) were separated from a 5 µL sample using a 5 mM ammonium acetate and 0.1% ammonium hydroxide aqueous solution as buffer A, and 100% acetonitrile as buffer B. The flow rate was 0.3 mL / min, and the column temperature was 60 °C. The method started with 70% buffer B for 0.2 min, then gradient-up to 100% B over 5.3 min, then restored to 70% B over 0.1 min, and equilibrated at 70% B for 3.9 min. The mass spectrometer was operated in negative ionization mode with an ion spray voltage of -4500 V. The source temperature was set to 450 °C, and the TOP mass range was 100–1000 m / z. The declustering potential was -120 V, and the collision energy was -10 V. The cumulative FFA concentration within the linear range was fitted with a linear regression to calculate the initial hydrolysis rate.

[0451] Host cell proteins in samples throughout the process were measured using an internal CHO protein (CHOP) ELISA assay, and DNA was quantified using an internal qPCR method.

[0452] Example 6

[0453] This example demonstrates a comparison between two purification platforms for antibody purification, which include: (1) deep filtration of HCCF using a 120ZB10A depth filter followed by protein A chromatography; or (2) deep filtration of HCCF using an EMPHAZE™ AEX depth filter followed by protein A chromatography.

[0454] Harvest cell culture medium (HCCF) was collected from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM3). For a purification platform with a 120 ZB10A depth filtration step (purification platform (1)), each HCCF pool was subjected to 120 ZB10A depth filtration (300 L / m³). 2Then, protein A chromatography was performed. Aliquots of the protein A-chromatographically separated aggregates were collected. The polysorbate hydrolytic activity of the aliquots was measured using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement rate of the aliquots after protein A chromatography relative to the FAMS rate is shown in Figure 12. Aliquots of the HCCF-chromatographically separated aggregates (before depth filtration) were used to obtain control measurements.

[0455] For purification platforms with an EMPHAZE™ AEX deep filtration step (purification platform (2)), each HCCF confluence was subjected to EMPHAZE™ AEX deep filtration (300 L / m³). 2 Then, protein A chromatography was performed. Aliquots of the protein A-chromatographically separated aggregates were collected. The polysorbate hydrolytic activity of the aliquots was measured using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement rate of the aliquots after protein A chromatography relative to the FAMS rate is shown in Figure 12. Aliquots of the HCCF-chromatographically separated aggregates (before depth filtration) were used to obtain control measurements.

[0456] Example 7

[0457] This example demonstrates a comparison between two purification platforms for antibody purification, which include: (1) protein A chromatography of HCCF followed by activated carbon (40CR) filtration followed by cation exchange chromatography using POROS® 50HS; or (2) protein A chromatography of HCCF followed by deep filtration using an X0SP depth filter followed by HIC using phenyl SEPHAROSE® rapid flow.

[0458] Harvest cell culture medium (HCCF) from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM4) was collected. For a purification platform without a deep filtration step (purification platform (1)), the three HCCF samples were subjected to protein A chromatography. The protein A chromatography pools were adjusted to pH 5.5 ± 0.3 with a tris(hydroxymethyl)aminomethane (Tris) base, and then the adjusted pools were filtered at 40 CR (300 L / m³). 2Then, POROS® 50 HS chromatography was performed. Aliquots of the merged product after 40CR filtration and aliquots of the merged product after POROS® 50HS filtration were collected. The polysorbate hydrolytic activity of the aliquots was measured using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement ratios of the aliquots of the merged product after 40CR filtration and the aliquots of the merged product after POROS® 50HS chromatography are shown in Figures 13 and 14, respectively. Control measurements were obtained using aliquots of the merged product chromatographically analyzed with Protein A (without 40CR filtration).

[0459] For the purification platform with the XOSP deep filtration step (purification platform (2)), the three HCCF samples were subjected to protein A chromatography. The protein A chromatography pools were adjusted to pH 5.5 ± 0.3 with a tris(hydroxymethyl)aminomethane (Tris) base, and then the adjusted pools were subjected to XOSP filtration (300 L / m³). 2 The mixture was then processed using phenyl SEPHAROSE® rapid flow chromatography. Aliquots of the XOSP-filtered and phenyl SEPHAROSE®-filtered mixtures were collected. The polysorbate hydrolytic activity of the aliquots was measured using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement ratios of the XOSP-filtered and phenyl SEPHAROSE®-filtered mixtures to the FAMS rate are shown in Figures 13 and 14, respectively. Control measurements were obtained using protein A chromatography aliquots (without XOSP depth filtration).

[0460] Example 8

[0461] This example demonstrates a comparison between three purification platforms for antibody purification, which include: (1) protein A chromatography of HCCF followed by activated charcoal (40CR) filtration and then multivariate chromatography using Capto Adhere; (2) protein A chromatography of HCCF followed by deep filtration using an EMPHAZE™ depth filter and then multivariate chromatography using Capto Adhere; or (3) protein A chromatography of HCCF followed by deep filtration using a PDD1 depth filter and then multivariate chromatography using Capto Adhere.

[0462] Harvest cell culture medium (HCCF) from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM4) was collected. For a purification platform without a deep filtration step (purification platform (1)), the three HCCF samples were subjected to protein A chromatography. The protein A chromatography pools were adjusted to pH 8.0 ± 0.5 with Tris base, and then the adjusted pools were filtered at 40 CR (300 L / m³). 2 Then, CaptoAdhere chromatography was performed. Aliquots of the merged mixture after 40 CR filtration were collected. The polysorbate hydrolytic activity of the aliquots was measured using LEAP, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement rate of the aliquots of the merged mixture after 40 CR filtration is shown in Figure 15. Control measurements were obtained using protein A chromatography of the aliquots of the merged mixture (without 40 CR filtration).

[0463] For the purification platform with the EMPHAZE™ deep filtration step (purification platform (2)), the three HCCF samples were subjected to protein A chromatography. The protein A chromatography pools were adjusted to pH 8.0 ± 0.5 with Tris base, and then the adjusted pools were subjected to EMPHAZE™ deep filtration (300 L / m³). 2 Then, Capto Adhere chromatography was performed. Aliquots of the merged mixture after EMPHAZE™ deep filtration were collected. The polysorbate hydrolytic activity of the aliquots was measured using LEAP assay, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement rate of the aliquots of the merged mixture after EMPHAZE™ deep filtration is shown in Figure 15. Control measurements were obtained using aliquots of the merged mixture chromatographically analyzed with Protein A (without EMPHAZE™ deep filtration).

[0464] For the purification platform with a PDD1 deep filtration step (purification platform (3)), the three HCCF samples were subjected to protein A chromatography. The protein A chromatography pool was adjusted to pH 8.0 ± 0.5 with Tris base, and then the adjusted pool was subjected to PDD1 deep filtration (300 L / m³). 2Then, Capto Adhere chromatography was performed. Aliquots of the merged mixture after PDD1 deep filtration were collected. The polysorbate hydrolytic activity of the aliquots was measured using LEAP, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measurement rate of the aliquots of the merged mixture after PDD1 deep filtration is shown in Figure 15. Control measurements were obtained using aliquots of the merged mixture (without PDD1 deep filtration) chromatographically analyzed with Protein A.

[0465] Example 9

[0466] This example demonstrates the evaluation and comparison of workflows for purifying TYRP1 TCB antibodies (such as those disclosed in PCT / EP2019 / 08614, which are incorporated herein by reference in their entirety). The workflows use different pre-C1 depth filters, followed by a pre-C2 filtration step using a Merck Millistak+® HC Pro X0SP filter. The pre-C1 depth filters compared are a depth filter containing chemically defined synthetic materials (3M™ EMPHAZE™ AEX Mix Purifier) ​​and a harvest clarification depth filter (ZETA PLUS™ EXT ZB Series, 120ZB). Both the 120ZB and EMPHAZE™ depth filters are positively charged, while the X0SP depth filter is negatively charged at pH > 4.5.

[0467] The experimental workflow and sample name assignment are shown in Figure 16. In short, a cell-free harvest fluid containing TYRP1 TCB antibody with approximately 80 NTU turbidity was collected from the bioreactor and used as the loading material for the experiment. Prior to protein A chromatography, a portion of the cell-free harvest fluid was filtered using a 120ZB depth filter, and a second portion was filtered using an EMPHAZE™ depth filter. Both filtrates were then sterile filtered and protein A chromatography was performed using MabSelect SuRe™ media from GE Healthcare. The eluents from the 120ZB depth filter workflow and the EMPHAZE™ depth filter workflow were titrated to pH 5.5 with 1 M TRIS / HCl (pH 9.0). For each workflow, the titrated eluent was aliquoted; one sample was sterilely filtered using a 0.2 µm sterile filter and retained as a reference, using a small Merck Millistak+® HC Pro X0SP filter (5 cm). 2Filter the second aliquot (approximately 100 mL to approximately 120 mL) using the filtration area. Collect three eluent fractions from the X0SP filter using the 120ZB workflow. Collect four eluent fractions from the X0SP filter using the EMPHAZE™ workflow. Each X0SP eluent fraction is then filtered using a 0.2 µm sterile filter.

[0468] Each aliquot was then analyzed by LEAP assay according to the method provided in Example 1. Comparison of the LEAP results showed that both pre-C1 depth filter workflows effectively reduced hydrolytic activity, and the X0SP filter significantly reduced the hydrolytic activity of the capture column output (Figure 17). A comparison of the 120ZB and EMPHAZE™ workflows showed that the EMPHAZE™ filter material exhibited 33% lower hydrolytic activity of protein A eluent compared to the 120ZB filter (Figure 17).

[0469] Example 10

[0470] This example demonstrates the evaluation and comparison of purification of different HCCF samples containing TYRP1 TCB antibodies using a purification platform (such as that disclosed in PCT / EP2019 / 08614, which is incorporated herein by reference in its entirety) that includes an XOSP deep filtration step for protein A chromatography eluents.

[0471] Two different HCCF samples (CF 238 and CF 239) were prepared from separate cultures of cells producing TYRP1 TCB antibodies. Protein A chromatography was performed using MabSelect SuRe™ media. The eluents (CF 238 MSS and CF 239 MSS) were collected, each titrated to pH 5.5 with 1 M TRIS / HCl (pH 9.0), and then subjected to XOSP depth filtration. Specifically, 35 μL of CF 238 MSS eluent or 20 μL of CF 239 MSS eluent was passed through an XOSP filter (1 mM, ... 2 or 0.55 m 2 ) at 160 L / m 2 Filtration was performed at a flow rate of / h. Meanwhile, aliquots of CF 238 and CF 239 HCCF samples were each purified using a reference purification platform excluding the X0SP depth filtration step following protein A chromatography.

[0472] Lipase activity was performed on each resulting eluate as discussed in Example 1. The lipase activity results obtained from the references used for both CF 238 and CF 239 and from the purification platform including the X0SP depth filter are shown in Figures 18A (CF 238) and 18B (CF 239). As shown in Figures 18A and 18B, the hydrolytic activity of the eluates from the purification platform including X0SP was significantly reduced.

[0473] Example 11

[0474] This example demonstrates a comparison of purification of pramizumab using four purification platforms that incorporate different depth filtration steps for filtering regulated affinity chromatography (protein A chromatography) eluents. Specifically, the four different depth filtration steps are based on: (i) PDD1; (ii) X0SP; (iii) PDD1 followed by X0SP; and (iv) X0SP followed by PDD1.

[0475] Adjust the affinity chromatography eluent to pH 6.0 + / - 0.2 with 2 M Tris. Equilibrate the PDD1 and XSOP filters with at least 220 mL of the appropriate buffer (25 mM Tris / acetate). Load both filters at < 200 L / m². Maintain a flow rate of 11 mL / min for both PDD1 and XSOP filters. Control the pressure during the experiment. Fractionate from the depth filter after 100 L / m² and 200 L / m².

[0476] Lipase activity and HCP levels in the protein A chromatography eluent from the reference method (no depth filtration; loading) were compared with eluents collected after each depth filtration step on the four purification platforms. Lipase activity was determined according to Example 1. The results of lipase activity in the fractions obtained from the depth filter are shown in Figure 19A. The results of HCP measurements are shown in Figure 19B.

Claims

1. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) Capture step; as well as (b) Depth filtering step, This reduces the rate of enzyme hydrolysis activity of the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step.

2. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) Capture step; as well as (b) Depth filtering step, This reduces the level of the hydrolytic enzyme in the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step.

3. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising: (a) Capture step; as well as (b) Depth filtering step, This reduces the degradation of the polysorbate in the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step.

4. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in the following order: (a) A capture step, the capture step comprising processing by affinity chromatography; (b) Virus inactivation steps; (c) Second polypeptide purification step; (d) The third polypeptide purification step; and (e) Ultrafiltration / difiltration (UFDF) steps, The purification platform further includes a depth filtration step performed on one or more of the following: (i) prior to the capture step; (ii) after the capture step and before the virus inactivation step; (iii) after the virus inactivation step and before the second polypeptide purification step; (iv) after the second peptide purification step and before the third peptide purification step; or (v) After the third peptide purification step and before the ultrafiltration / percolation (UFDF) step; This reduces the rate of enzymatic hydrolysis activity of the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step.

5. A pharmaceutical composition obtained by the method according to any one of claims 1-4.

6. A formulated antibody moiety composition comprising an antibody moiety and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, and wherein the composition has a shelf life of more than 24 months.

7. A method for reducing the rate of enzymatic hydrolysis of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in sequence: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A purification step, which includes treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography. The purification platform further includes one or more depth filtration steps. The one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step. Each depth filtering step includes processing via a depth filter, and The depth filter described herein comprises materials selected from the group consisting of: (i) Silica and polyacrylic acid fibers; (ii) Hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; as well as (iii) Cellulose fibers, diatomaceous earth and perlite This reduces the rate of enzymatic hydrolysis activity of the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

8. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in sequence: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A purification step, which includes treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography. The purification platform further includes one or more depth filtration steps. The one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step and before the purification step; or after the purification step. Each depth filtering step includes processing via a depth filter, and The depth filter described herein comprises materials selected from the group consisting of: (i) Silica and polyacrylic acid fibers; (ii) Hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; as well as (iii) Cellulose fibers, diatomaceous earth and perlite This reduces the level of one or more hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

9. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in sequence: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A purification step, which includes treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-component chromatography. The purification platform further includes one or more depth filtration steps. The one or more depth filtration steps are performed at any one or more of the following times: before the capture step; after the capture step; or after the capture step and before the purification step. Each depth filtering step includes processing via a depth filter, and The depth filter described herein comprises materials selected from the group consisting of: (i) Silica and polyacrylic acid fibers; (ii) Hydrogel Q (quaternary ammonium)-functionalized nonwoven media and multi-zone microporous membranes; as well as (iii) Cellulose fibers, diatomaceous earth and perlite This reduces the degradation of polysorbate in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps mentioned above.

10. A pharmaceutical composition obtained by the method according to any one of claims 7-9.

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