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

Abstract

The present disclosure provides purification platforms comprising a depth filtration step and / or a hydrophobic interaction chromatography (HIC) step. Also disclosed herein are methods of using the purification platforms described herein and compositions, such as pharmaceutical compositions, obtained from the purification platforms.

Description

[0001] Cross Reference to Related Applications

[0002] 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 each of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure provides purification platforms comprising a depth filtration step and / or a hydrophobic interaction chromatography (HIC) step. Also disclosed herein are methods of using the purification platforms described herein and compositions obtained from the purification platforms. BACKGROUND

[0004] Biological therapeutic products produced from host cell cultures, such as antibodies, require purification to remove host cell proteins and other impurities that can affect, for example, product quality and therapeutic efficacy. Current purification methods can not remove all host cell proteins and impurities, including host cell hydrolytic enzymes. Thus, host cell proteins and impurities that remain with the purified target can affect the purified target itself as well as other additives, e.g., components added for formulation purposes, such as surfactants. Accordingly, there is a need for improved methods for purifying biological therapeutic products produced from host cell cultures for pharmaceutical use.

[0005] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. SUMMARY

[0006] In one aspect, a method of reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform comprising: (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.

[0007] In some embodiments, the rate of enzymatic hydrolysis activity is the rate of polysorbate ester hydrolysis activity. In some embodiments, the relative reduction in the rate of enzymatic 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.

[0008] In another aspect, a method of reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform comprising: (a) a capture step; and (b) a depth filtration step, thereby reducing the level of hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the depth filtration step. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate esters. 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 the depth filtration step is at least about 20%.

[0009] In another aspect, a method for reducing degradation of polysorbate esters in a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform comprising: (a) a capture step; and (b) a depth filtration step, thereby reducing 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 degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without the depth filtration step is at least about 5%.

[0010] In some embodiments, the purification platform is used to purify a target from a sample, wherein the sample comprises the target and one or more host cell impurities. In some embodiments, the target comprises a polypeptide. In some embodiments, the host cell impurity is a host cell protein.

[0011] In some embodiments, the depth filtration step is performed prior to the capture step, or the depth filtration step is performed after the capture step.

[0012] In some embodiments, the depth filtration step comprises processing 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, a cellulose fiber, a polymer fiber, a tacky resin, and an ash composition. In some embodiments, at least a portion of the substrate of the depth filter comprises a surface modification. In some embodiments, the surface modification is one or more of a quaternary amine surface modification, a cationic surface modification, and an anionic surface modification. In some embodiments, the depth filter is selected from the group consisting of an EMPHAZE® depth filter, an X0SP depth filter, a PDD1 depth filter, a ZETA PLUS® 120ZA depth filter, and a ZETA PLUS® 120ZB depth filter. TM In some embodiments, the depth filtration step comprises processing 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, a cellulose fiber, a polymer fiber, a tacky resin, and an ash composition. In some embodiments, at least a portion of the substrate of the depth filter comprises a surface modification. In some embodiments, the surface modification is one or more of a quaternary amine surface modification, a cationic surface modification, and an anionic surface modification. In some embodiments, the depth filter is selected from the group consisting of an EMPHAZE® depth filter, an X0SP depth filter, a PDD1 depth filter, a ZETA PLUS® 120ZA depth filter, and a ZETA PLUS® 120ZB depth filter. TM 120ZA depth filter, and a ZETA PLUS® 120ZB depth filter. TM 120ZA depth filter, and a ZETA PLUS® 120ZB depth filter.

[0013] 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.

[0014] 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.

[0015] In some embodiments, the purification platform further includes another depth filtration step performed prior to the capture step.

[0016] 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.

[0017] 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.

[0018] 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 the UFDF step, if present.

[0019] In some embodiments, 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.

[0020] 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.

[0021] In some embodiments, the HIC purification step includes processing through a HIC filter.

[0022] In some embodiments, each of the one or more purification steps independently comprises processing by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge induction chromatography, ceramic hydroxyapatite chromatography, and multimodal chromatography. In some embodiments, each of the one or more purification steps independently comprises processing by chromatography selected from the group consisting of: DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel TM , Capto MMC, and Capto Adhere.

[0023] In another aspect, a method of reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform comprising, in the following order: (a) a capture step comprising processing by affinity chromatography; (b) a viral inactivation step; (c) a second polypeptide purification step; (d) a third polypeptide purification step; and (e) an ultrafiltration / diafiltration (UFDF) step, wherein the purification platform further comprises a depth filtration step performed upon one or more of: (i) prior to the capture step; (ii) after the capture step and prior to the viral inactivation step; (iii) after the viral inactivation step and prior to the second polypeptide purification step; (iv) after the second polypeptide purification step and prior to the third polypeptide purification step; or (v) after the third polypeptide purification step and prior to the ultrafiltration / diafiltration (UFDF) 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.

[0024] In some embodiments, the purification platform further comprises, in the following order, a pH hold step and a viral filtration step performed after the third polypeptide purification step and prior to the UFDF step. In some embodiments, the viral filtration step comprises processing by a viral filter.

[0025] In some embodiments, the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step performed upon one or more of: (i) after the third polypeptide purification step and prior to the pH hold step; (ii) after the pH hold step and prior to the viral filtration step; or (iii) after the viral filtration step and prior to the UFDF step.

[0026] In some embodiments, the method further comprises determining the rate of enzymatic hydrolysis activity of the composition.

[0027] In some embodiments, the method further comprises determining the level of one or more hydrolytic enzymes in the composition.

[0028] 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.

[0029] In some embodiments, the method further comprises a sample processing step.

[0030] In some embodiments, the sample is or is derived from a cell culture sample. In some embodiments, the cell culture sample comprises a host cell, and wherein the host cell is a Chinese hamster ovary (CHO) cell or an E. coli cell. In some embodiments, the sample comprises a host cell or a component derived from the host cell. In some embodiments, the sample comprises one or more host cell proteins, and wherein one of the one or more host cell proteins is a hydrolase.

[0031] In some embodiments, the hydrolase is a lipase, an esterase, a thioesterase, a phospholipase, or a ceramidase.

[0032] In some embodiments, the sample comprises a target, and 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.

[0033] In some embodiments, the antibody moiety is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0034] In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0035] In another aspect, a pharmaceutical composition obtained from any of the methods described herein is provided.

[0036] In another aspect, a formulated antibody moiety composition comprising a polysorbate is provided, wherein the composition has a reduced rate of polysorbate hydrolytic activity, and wherein the shelf life of the composition exceeds 24 months.

[0037] In another aspect, a formulated antibody moiety composition comprising an antibody moiety and a polysorbate is provided, wherein the composition has a reduced rate of polysorbate hydrolytic activity, wherein the shelf life of the composition is extended compared to the shelf life indicated in a document submitted to a health authority in connection with the formulated antibody moiety composition, and wherein the shelf life is extended by at least 6 months compared to the shelf life indicated in the document.

[0038] In another aspect, a formulated antibody moiety composition comprising an antibody moiety is provided, wherein the formulated antibody moiety composition has reduced polysorbate degradation, wherein the degradation is reduced by at least about 20% compared to the degradation indicated in a document submitted to a health authority in connection with the formulated antibody moiety composition.

[0039] In another aspect, a formulated antibody moiety composition comprising an antibody moiety and a polysorbate is provided, wherein the polysorbate degrades 20% or less per year during storage of the liquid composition.

[0040] In some embodiments, the antibody moiety of the formulated antibody moiety composition is a monoclonal antibody. In some embodiments, the antibody moiety of the formulated antibody moiety composition is a human antibody, a humanized antibody, or a chimeric antibody.

[0041] In some embodiments, the antibody moiety of the formulated antibody moiety composition is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0042] In some embodiments, the antibody moiety of the formulated antibody moiety composition is selected from the group consisting of: ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lexibulinab, omalizumab, ranibizumab, emicizumab, seruplinab, prasinezumab, RO6874281, and RO7122290.

[0043] In some embodiments, the polysorbate hydrolytic activity rate of the formulated antibody moiety 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.

[0044] In another aspect, a method of reducing the enzymatic hydrolytic activity rate of a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform, the purification platform comprising, in sequence: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step; or after the capture step and prior to the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the enzymatic hydrolytic activity rate 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 enzymatic hydrolytic activity rate is an enzyme polysorbate hydrolytic activity rate. In some embodiments, the relative reduction in the enzymatic hydrolytic activity rate of the composition compared to purifying the sample using the same purification platform without the depth filtration step is at least about 20%.

[0045] In another aspect, a method of reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform, the purification platform comprising, in sequence: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step and prior to the purification step; or after the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing 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. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate esters. 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 depth filtration step.

[0046] In another aspect, a method of reducing degradation of polysorbate esters in a composition obtained from a purification platform is provided, the method comprising subjecting a sample to a purification platform, the purification platform comprising, in sequence: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step; or after the capture step and prior to the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing 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 esters in the composition is at least about 5% compared to purifying the sample using the same purification platform without the depth filtration step.

[0047] In some embodiments, the depth filter comprising silica and polyacrylic fibers comprises a silica filter aid and a polyacrylic fiber pulp.

[0048] In some embodiments, the depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane comprises four layers comprising a hydrogel Q- functionalized nonwoven material and a nine-zone microporous membrane.

[0049] In some embodiments, 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 resinous binder.

[0050] In some embodiments, the depth filter is selected based on the pH of the solution entering the depth filter. In some embodiments, the depth filter comprising silica and polyacrylic fibers is selected when the solution entering the depth filter is from about 5 to about 6.5. In some embodiments, the depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane is selected when the solution entering the depth filter is from about 7 to about 8.5. In some embodiments, the method further comprises selecting the depth filter based on the pH of the solution entering the depth filter.

[0051] In some embodiments, the purification platform comprises, in sequence: a depth filtration step comprising processing through a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane; a capture step comprising processing through protein A chromatography; and a purification step.

[0052] In some embodiments, the purification step comprises processing through HIC. In some embodiments, the HIC is phenyl fast flow chromatography.

[0053] In some embodiments, the purification step comprises processing through cation exchange chromatography. In some embodiments, the cation exchange chromatography is Capto S. 50 HS.

[0054] In some embodiments, the purification platform further comprises a second depth filtration step comprising processing through a depth filter comprising silica and polyacrylic fibers, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0055] In some embodiments, the purification step comprises processing through multi-modal chromatography. In some embodiments, the multi-modal chromatography is Capto Adhere.

[0056] In some embodiments, the purification platform further comprises a second depth filtration step comprising processing through a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0057] In some embodiments, the purification platform is used to purify a target from a sample, wherein the sample comprises the target and one or more host cell impurities. In some embodiments, the target comprises a polypeptide. In some embodiments, the host cell impurity is a host cell protein.

[0058] In some embodiments, the purification platform further comprises a viral inactivation step, wherein the viral inactivation step occurs after the capture step. In some embodiments, the one or more depth filtration steps occur after the viral inactivation step.

[0059] In some embodiments, the purification platform further comprises an ultrafiltration / diafiltration (UFDF) step, and wherein the UFDF step occurs after the purification step.

[0060] In some embodiments, the methods described herein further comprise determining the rate of enzymatic hydrolysis activity of the composition.

[0061] In some embodiments, the methods described herein further comprise determining the level of one or more hydrolytic enzymes in the composition.

[0062] 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.

[0063] In some embodiments, the methods described herein further comprise a sample processing step.

[0064] In some embodiments, the sample is or is derived from a cell culture sample. In some embodiments, the cell culture sample comprises a host cell, and wherein the host cell is a Chinese hamster ovary (CHO) cell or an E. coli cell. In some embodiments, the sample comprises a host cell or a component derived from the host cell.

[0065] 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: olizumab, pertuzumab, trastuzumab, tocilizumab, faliximab, polotuzumab, gantinguzumab, cybituzumab, crorezumab, mosunituzumab, tirelumab, bevacizumab, rituximab, atezolizumab, olizumab, lanperizumab, lejinzumab, omalizumab, lannizumab, emecizumab, celuzumab, prasinizumab, RO6874281, and RO7122290.

[0066] On the other hand, a pharmaceutical composition obtained from any of the methods described herein is provided.

[0067] 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

[0068] Figure 1A Exemplary steps of the purification platform 100 are shown. Figure 1B Exemplary options for the steps used to purify the platform are shown.

[0069] Figure 2 A bar graph showing the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS is presented.

[0070] Figure 3 A bar graph showing the hydrolytic activity measured in the composition obtained from the purification platform using lipase activity assay is presented.

[0071] Figure 4 A bar graph showing the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS is presented.

[0072] Figure 5 A schematic diagram of the purification options for faliximab is shown.

[0073] Figure 6A and Figure 6B This shows the result before filtering with the PDD1 filter ( Figure 6A ) and afterwards ( Figure 6B Bar graph of PS20 hydrolytic activity measured in the FcXL eluent of faliximab.

[0074] Figure 7 A bar graph showing the amount of free fatty acids measured in the composition obtained from the purification platform using FAMS is presented.

[0075] Figure 8A to Figure 8B The bar graph shows the hydrolytic activity measured in the protein A eluate after filtration using a depth filter.

[0076] Figure 9 EMPHAZE was shown. TM The relative levels of CHOP and polysorbate degradation activity in the protein A eluate after deep filter clarification.

[0077] Figure 10 The specific activity of the composition obtained from the X0SP depth filter (pH 5.5) for polysorbate degradation at different fluxes is shown.

[0078] Figure 11A to Figure 11C The results show that olizumab at pH 5.5 ( Figure 11A ), at pH 5.5, celumab ( Figure 11B ) and tocilizumab at pH 6.5 ( Figure 11C ) Specific activity of polysorbate degradation at different fluxes.

[0079] Figure 12 A bar graph showing the specific FAMS rate of the composition obtained from the purification platform is presented.

[0080] Figure 13 A bar graph showing the specific FAMS rate of the composition obtained from the purification platform is presented.

[0081] Figure 14 A bar graph showing the specific FAMS rate of the composition obtained from the purification platform is presented.

[0082] Figure 15 A bar graph showing the specific LEAP rate of compositions obtained from the purification platform.

[0083] Figure 16 A schematic of the purification workflow is shown.

[0084] Figure 17 A bar graph showing the average conversion of compositions obtained from the purification platform measured using the LEAP assay.

[0085] Figure 18A and Figure 18B A bar graph showing the hydrolytic activity of compositions obtained from the purification platform measured using the lipase activity assay. Figure 18A Results obtained from CF 238 are shown. Figure 18B Results obtained from CF 239 are shown.

[0086] Figure 19A A bar graph showing the average conversion of compositions obtained from the purification platform measured using the LEAP assay. Figure 19B A bar graph showing the hydrolytic activity of compositions obtained from the purification platform measured using the lipase activity assay. DETAILED DESCRIPTION

[0087] In some aspects, the present application provides methods for purifying a target from a sample comprising the target, the methods comprising subjecting the sample to a purification platform disclosed herein, the purification platform comprising one or more depth filtration steps and / or one or more hydrophobic interaction chromatography (HIC) steps.

[0088] The present disclosure is based in part on the unexpected finding that purification platforms comprising one or more depth filtration steps, such as depth filtration steps performed on host cell culture fluid (HCCF) and / or affinity chromatography eluate, reduce the rate of enzymatic hydrolysis activity of compositions obtained therefrom. In addition, the present disclosure is based in part on the unexpected finding that purification platforms comprising one or more HIC steps reduce the rate of enzymatic hydrolysis activity of compositions obtained therefrom, and purification platforms comprising both depth filtration steps and HIC steps can further reduce the rate of enzymatic hydrolysis activity of compositions obtained therefrom.

[0089] Those skilled in the art will also appreciate that modifications can be made to the forms and details of the implementations described herein without departing from the scope of the disclosure. Additionally, although various advantages, aspects, and objects of the disclosure have been described above in specificity, the scope of the disclosure should not be construed as limited to the advantages, aspects, and objects described herein, but instead, are to cover all obtaining or making or using the principles and novel implementations of the disclosure within the true scope of the disclosure.

[0090] DEFINITIONS

[0091] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, singular forms of words shall be construed as including the plural and vice versa. If any definition set forth herein conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0092] The term“antibody portion” includes full-length antibodies and antigen-binding fragments thereof. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both the light and heavy chains typically comprise three hypervariable 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 antibodies and antigen-binding fragments disclosed herein can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are flanked by stretches of sequences called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequences of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, d, e, g, and m heavy chains, respectively. Several of the major antibody classes are divided into subclasses, e.g., lgG1 (g1 heavy chain), lgG2 (g2 heavy chain), lgG3 (g3 heavy chain), lgG4 (g4 heavy chain), lgA1 (a1 heavy chain), or lgA2 (a2 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 diabody. 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 an interleukin. In some embodiments, the antibody portion is linked to a protein that facilitates entry across the blood-brain barrier.

[0093] As used herein, the term "antigen binding fragment" refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelid-derived single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen binding fragment is capable of binding to the same antigen bound by the parent antibody or parent antibody fragment, e.g., a parent scFv. In some embodiments, an antigen binding fragment can comprise one or more CDRs from a particular human antibody grafted to framework regions from one or more different human antibodies.

[0094] The term "chimeric antibody" refers to an antibody in which some part of the heavy and / or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the biological activity of the present application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).

[0095] As used herein, the term "multispecific antibody" refers to a monoclonal antibody that has binding specificities for at least two different sites (i.e., different antigens or different epitopes on the same antigen). In certain aspects, the multispecific antibody has two binding specificities (a bispecific antibody). In certain aspects, the multispecific antibody has three or more binding specificities. The multispecific antibody can be prepared as a full-length antibody or an antibody fragment.

[0096] The term "semisynthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.

[0097] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains arises the three antigen- binding loops (three loops from the VH and three loops from the VL) that contribute the amino acid residues for binding of antigens. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0098] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0099] The term "diabodies" refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) in which the VHand VLdomains are typically joined by a short linker (such as about 5 to about 10 residues) so that they form a dimeric protein with two antigen- binding sites, i.e., a bivalent fragment. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VHand VLdomains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0100] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human antibodies. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired antibody specificity, affinity, and function. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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).

[0101] In some embodiments, the methods described herein include one or more depth filtration steps. A depth filtration step is a type of chromatography that includes processing through a depth filter. In some embodiments, the depth filter comprises a porous filtration medium capable of retaining portions of the sample such as cellular components and debris, where filtration occurs, for example, within the depth of the filter material. In some embodiments, the depth filter comprises a synthetic material, a non-synthetic material, or a combination thereof. In some embodiments, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, a cellulose fiber, a polymer fiber, a tacky resin, and an ash composition. In some embodiments, at least a portion of the substrate of the depth filter includes a surface modification. In some embodiments, the surface modification is one or more of a quaternary amine surface modification, a cationic surface modification, and an anionic surface modification. In some embodiments, the depth filter is selected from the group consisting of EMPHAZE® TM depth filters such as EMPHAZE® TM AEX depth filters, X0SP depth filters, PDD1 depth filters, ZETA PLUS TM 120ZA depth filters, and ZETA PLUS TM 120ZB depth filters.

[0102] 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 a filter aid comprising one or more of diatomaceous earth or perlite, and wherein each layer further comprises a resin binder. In some embodiments, the depth filter is a PDD1 depth filter.

[0103] 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 comprise diatomaceous earth and / or perlite. In some embodiments, the depth filter is an XOSP depth filter.

[0104] 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% silanols that ionize at pH 7. In some embodiments, the silica filter aid is a silica gel filter aid wherein about 50% of the silanols in the silica filter aid ionize at pH 7. In some embodiments, the silica filter aid is derived from silica (such as silica gel). (Evonik Industries AG) or silica gel (such as Kieseigel 60 (Merck KGaA)) precipitation. In some embodiments, the polyacrylic fibers are nonwoven polyacrylic fiber pulp. In some embodiments, the polyacrylic fibers are electrospun polyacrylic nanofibers. In some embodiments, the degree of fibrillation of the polyacrylic fibers 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 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 1.5m 2 Such as approximately 0.11m 2 Approximately 0.55m 2 or about 1.1m 2In some embodiments, the depth filter does not comprise diatomaceous earth and / or perlite. In some embodiments, the depth filter comprises two layers of filtration media, wherein the first layer comprises a silica filter aid having about 50% silanol ionized at pH 7, and the second layer comprises a polyacrylic fiber pulp having a degree of polyacrylic fiber fibrillation associated with a Canadian Standard Freeness (CSF) of about 10 mL to about 800 mL, and wherein the depth filter does not comprise diatomaceous earth.

[0105] In some embodiments, the depth filter comprises a hydrogel Q (quaternary amine)- 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 does not comprise diatomaceous earth and / or perlite. In some embodiments, the depth filter is an EMPHAZE® depth filter. TM AEX depth filter.

[0106] In some embodiments, the depth filter comprises a plurality of components or layers. In some embodiments, the depth filter comprises a plurality of layers comprising one or more layers comprising anion exchange (AEX) functional polymers. In some embodiments, the layer comprising AEX functional polymers comprises quaternary ammonium (Q), such as Q-functional hydrogels. In some embodiments, the layer comprising AEX functional polymers comprises quaternary ammonium (Q) functional polymers associated with a nonwoven article. In some embodiments, the layer comprising AEX functional polymers comprises quaternary ammonium (Q) functional hydrogels covalently grafted to a fine fiber polypropylene nonwoven scaffold. In some embodiments, the depth filter comprises a plurality of layers comprising a layer comprising a multi-zone membrane comprising a nine-zone membrane having a pore size of about 0.05 pm to about 0.3 pm, such as about 0.22 pm. In some embodiments, the depth filter does not comprise diatomaceous earth.

[0107] In some embodiments, the methods described herein comprise one or more hydrophobic interaction chromatography (HIC) steps. A HIC step is a type of chromatography that involves processing through a HIC medium, such as a HIC filter or a HIC column. In some embodiments, the HIC medium comprises a hydrophobic moiety comprising, for example, a methyl, ethyl, propyl, octyl, or phenyl group. In some embodiments, the sample is applied to the HIC medium in a polar buffer. In some embodiments, the polypeptide is eluted from the HIC medium using step elution with aqueous buffers having a gradually decreasing salt concentration, a gradually increasing detergent concentration, and / or a pH adjustment.

[0108] In some embodiments, the methods described herein are capable of reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform. In some embodiments, the rate of enzymatic hydrolysis activity represents the rate of activity of one or more hydrolytic enzymes, such as one or more different hydrolytic enzymes. In some embodiments, the rate of enzymatic hydrolysis activity is a surrogate measure of the activity of one or more enzymes in the composition. In some embodiments, the rate of enzymatic hydrolysis activity is measured by a surrogate substrate. In some embodiments, the rate of enzymatic hydrolysis activity is assessed by measuring a hydrolysis product of one or more hydrolytic enzymes.

[0109] As used herein, the terms “comprising,” “having,” “including,” and “containing,” and other similar forms, as well as grammatical equivalents thereof, are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, an article that is “comprising” component (element) A, B, and C can consist of (i.e., contain only) component (element) A, B, and C, or can contain not only component (element) A, B, and C, but also one or more other components (elements). Thus, “comprising” and like forms are intended to be inclusive or open-ended and to cover embodiments of the disclosure that “consist of’ or “consist essentially of,” in addition to those that “comprise.”

[0110] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0111] Reference to “about” an amount or parameter herein includes (and describes) variations that are directed to that amount or parameter per se. For example, description referring to “about X” includes description of “X”.

[0112] As used herein (including the appended claims), the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0113] purification platform

[0114] In some aspects of the disclosure, a purification platform comprising a depth filtration step and / or a hydrophobic interaction chromatography (HIC) step is provided. In some embodiments, a purification platform represents a workflow for purifying a target to any extent from a sample comprising the target. In some embodiments, the process workflow of a purification platform is the order of steps involved in purifying a target from a sample comprising the target.

[0115] For the purposes of example and explanation of the content disclosed herein, Figure 1A The sequential workflow of a portion of an exemplary purification platform 100 is illustrated below. Figure 1A As shown, 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, Figure 1A The exemplary purification platform shown includes one or more depth filtration steps. In some embodiments, Figure 1A The exemplary purification platform shown includes a deep filtration step performed after conditioning step 110 and before one or more purification steps. In some embodiments, Figure 1A The exemplary purification platform shown includes a deep filtration step prior to capture step 105. In some embodiments, Figure 1A The exemplary purification platform shown 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, Figure 1A The exemplary purification platform shown includes one or more depth filtration steps and one or more HIC steps.

[0116] 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 covered by the disclosure of the individual components (assemblies) and / or their steps.

[0117] Deep filtering steps

[0118] In some aspects, the present disclosure provides a purification platform comprising a depth filtration step. As described herein, the depth filtration step can be placed in any of one or more positions within the purification platform. In some embodiments, the purification platform described herein comprises one or more depth filtration steps at any stage of the process workflow, such as any of 2, 3, 4, or 5 depth filtration steps. In some embodiments, wherein the purification platform comprises more than one depth filtration step, the depth filtration steps are not performed in direct sequence, i.e., without some intervening step of the purification platform between the depth filtration steps. In some embodiments, wherein the purification platform comprises more than one depth filtration step, the depth filtration steps are the same. In some embodiments, wherein the purification platform comprises more than one depth filtration step, the depth filtration steps are different, e.g., comprising use of different depth filters.

[0119] In some embodiments, wherein the purification platform comprises more than one depth filtration step, a first depth filtration step occurs before a capture step comprising processing by protein A chromatography, and a second depth filtration step occurs after the capture step and before a purification step.

[0120] In some embodiments, the depth filtration step comprises processing by a depth filter. In some embodiments, the depth filter is a depth filter comprising a synthetic material. Depth filtration steps, including steps involved in processing by a depth filter, are known in the art. See, e.g., Yigzaw et al., Biotechnol Prog, 22, 2006 and Liu et al., mAbs, 2, 2010, which are hereby incorporated by reference in their entireties. One of ordinary skill in the art will appreciate, e.g., components (assemblies), conditions, and reagents involved in a depth filtration step.

[0121] In some embodiments, the methods described herein include one or more depth filtration steps, each depth filtration step comprising processing through a depth filter, wherein the depth filter is selected based on the pH of the solution entering the depth filter. In some embodiments, a depth filter comprising silica and polyacrylic fibers, such as a X0SP depth filter, is selected when the solution entering the depth filter is from about 5 to about 6.5. In some embodiments, a depth filter comprising silica and polyacrylic fibers, such as a X0SP depth filter, is selected when the solution entering the depth filter is about 6.5 or less, such as any of 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. In some embodiments, a depth filter comprising silica and polyacrylic fibers, such as a X0SP depth filter, is selected when the solution entering the depth filter is about any of 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. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is from about 7 to about 8.5. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about 8.5 or less, such as any of about 8.4 or less, 8.3 or less, 8.2 or less, 8.1 or less, 8.0 or less, 7.9 or less, 7.8 or less, 7.7 or less, 7.6 or less, 7.5 or less, 7.4 or less, 7.3 or less, 7.2 or less, or 7.1 or less. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, or 7.1. TM In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about 7 or more, such as any of about 7.1 or more, 7.2 or more, 7.3 or more, 7.4 or more, 7.5 or more, 7.6 or more, 7.7 or more, 7.8 or more, 7.9 or more, 8.0 or more, 8.1 or more, 8.2 or more, 8.3 or more, 8.4 or more, or 8.5 or more. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about any of 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. TM In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about 7 or more, such as any of about 7.1 or more, 7.2 or more, 7.3 or more, 7.4 or more, 7.5 or more, 7.6 or more, 7.7 or more, 7.8 or more, 7.9 or more, 8.0 or more, 8.1 or more, 8.2 or more, 8.3 or more, 8.4 or more, or 8.5 or more. In some embodiments, a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane, such as an EMPHAZE® depth filter, is selected when the solution entering the depth filter is about any of 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. TMDepth Filter. In some embodiments of the methods described herein, the method may further include selecting a 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 characteristics 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.

[0122] 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.

[0123] 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.

[0124] In some embodiments, the depth filter is selected from EMPHAZE. TM Depth filters (such as EMPHAZE) TM AEX depth filter, X0SP depth filter, PDD1 depth filter, ZETA PLUS TM 120ZA depth filter and ZETAPLUS TM A group consisting of 120ZB depth filters.

[0125] HIC steps

[0126] 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.

[0127] In some embodiments, the HIC step comprises processing through a HIC medium, such as a HIC column or a HIC membrane. HIC steps, including steps involved in processing through a HIC medium, are known in the art. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference. One of ordinary skill in the art will appreciate, e.g., components (assemblies), conditions, and reagents involved in HIC steps.

[0128] In some embodiments, the HIC medium comprises a hydrophobic resin. In some embodiments, at least a portion of the substrate of the HIC medium comprises a surface modification. In some embodiments, the surface modification is a phenyl or butyl surface modification.

[0129] In some embodiments, the HIC step is a flow-through mode HIC step. In some embodiments, the HIC step is a bind-and-elute mode HIC step.

[0130] In some embodiments, the purification platform comprises 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.

[0131] Capture step

[0132] In some embodiments, the purification platform comprises a capture step. In some embodiments, the capture step comprises processing through affinity chromatography.

[0133] Capture steps, including steps involved in processing through, e.g., affinity chromatography, are known in the art. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference.

[0134] In some embodiments, the 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, kappa chromatography, and kappa XL chromatography. In some embodiments, the capture step comprises processing through protein A chromatography. In some embodiments, the capture step comprises processing through FcXL chromatography.

[0135] In some embodiments, the protein A chromatography is silica-based protein A chromatography. In some embodiments, the protein A chromatography is agarose-based protein A chromatography. In some embodiments, the protein A chromatography is organic polymer-based protein A chromatography.

[0136] In some embodiments, the protein A chromatography is selected from the group consisting of ProA® vA TM 、 ProA® vA Ultra, ProA® Protein A fast flow, MabSelect TM , MabSelect TM SuRe, A and MabCapture TM consisting of.

[0137] conditioning step

[0138] In some embodiments, the purification platform includes a conditioning step. In some embodiments, the conditioning step is performed after the capture step.

[0139] Conditioning steps (including steps involved in processing of conditioning steps) are known in the art. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference.

[0140] In some embodiments, the conditioning step includes a viral inactivation step, such as a low pH hold step. In some embodiments, the low pH hold step is performed at a pH of about 2.5 to about 4. In some embodiments, the low pH hold step is configured for viral inactivation. In some embodiments, the low pH hold step is capable of inactivating endogenous / exogenous viruses.

[0141] one or more purification steps

[0142] 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 step and the conditioning step. In some embodiments, the one or more purification steps include a polypeptide purification step. In some embodiments, the one or more purification steps include more than one (such as any of 2, 3, 4, or 5) polypeptide purification steps.

[0143] Polypeptide purification steps (including steps involved in processing of polypeptide purification steps) are known in the art. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference.

[0144] In some embodiments, the polypeptide purification step includes processing by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induction chromatography, ceramic hydroxyapatite chromatography, and multimodal chromatography.

[0145] In some embodiments, the polypeptide purification step includes processing by chromatography selected from the group consisting of: diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), trimethylaminoethyl (TMAE), quaternary amine, quaternary aminoethyl (QAE), sulfopropyl (SP), SP- fast flow (FF), SP- XL, quaternary amine (Q) FF, mercaptoethylpyridine (MEP) - Hypercel TM Capto MMC (multimodal chromatography), Capto Adhere, XS and 50HS.

[0146] In some embodiments, the polypeptide purification step is a bind and elute polypeptide purification step. In some embodiments, the polypeptide purification step is a flow-through polypeptide purification step. In some embodiments, the polypeptide purification step is a weak partition chromatography polypeptide purification step. In some embodiments, the polypeptide purification step is an overload polypeptide purification step.

[0147] virus filtration step

[0148] In some embodiments, the purification platform includes a virus filtration step. In some embodiments, the virus filtration step is performed after the one or more purification steps.

[0149] The virus filtration step (including steps involved in the processing of the virus filtration step) is known in the art. See, e.g., Liu et al. mAbs, 2, 2010 and U.S. Application No. 20140309403, which are hereby incorporated by reference.

[0150] In some embodiments, the virus filtration step includes processing through a virus filter. In some embodiments, the virus filtration step includes a pH hold step. In some embodiments, the processing through a virus filter is performed after the pH hold step.

[0151] UFDF step

[0152] 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 filtration step.

[0153] The UFDF step (including steps involved in the processing of the UFDF step) is known in the art. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference.

[0154] In some embodiments, the UFDF step includes processing through ultrafiltration. In some embodiments, the UFDF step is performed in tangential flow filtration (TFF) mode. In some embodiments, the UFDF step includes processing through tangential flow filtration (such as high performance tangential flow filtration).

[0155] As described above, the purification platforms disclosed in the present application can 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 depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step. In some embodiments, the purification platform further includes a second depth filtration step. In some embodiments, the purification platform further includes a HIC step.

[0156] In some embodiments, the purification platform includes, in order: a capture step; and a conditioning step, wherein the purification platform further includes a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step. In some embodiments, the purification platform further includes a second depth filtration step. In some embodiments, the purification platform further includes a HIC step.

[0157] In some embodiments, the purification platform includes, in order: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further includes a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the purification platform further includes a second depth filtration step, such as a depth filtration step performed prior to 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.

[0158] In some embodiments, the purification platform comprises, in order: a capture step; a conditioning step; one or more purification steps; and a virus filtration step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the virus filtration step. In some embodiments, the depth filtration step is performed after the virus filtration step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as one or more HIC steps selected from being performed after the one or more purification steps and / or after the pH hold step of the virus filtration step, such as after the virus filtration step or during the virus filtration step.

[0159] In some embodiments, the purification platform comprises, in order: a capture step; a conditioning step; one or more purification steps; and a UFDF step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the UFDF step. In some embodiments, the depth filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as one or more HIC steps selected from being performed after the one or more purification steps and / or after the UFDF step.

[0160] In some embodiments, the purification platform comprises, in order: a capture step; a conditioning step; one or more purification steps; a virus filtration step; and a UFDF step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the virus filtration step. In some embodiments, the depth filtration step is performed after the virus filtration step and prior to the UFDF step. In some embodiments, the depth filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as one or more HIC steps selected from a pH hold step after the one or more purification steps and / or the virus filtration step, a HIC step after or during the virus filtration step, such as after and / or after the UFDF step.

[0161] In some embodiments, the purification platform comprises, in order: 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 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.

[0162] In some embodiments, the purification platform comprises, in order: 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.

[0163] In some embodiments, the purification platform comprises, in sequence: a capture step; a conditioning step; and one or more purification steps, 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0164] In some embodiments, the purification platform comprises, in sequence: a capture step; a conditioning step; one or more purification steps; and a viral filtration 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and prior to the viral filtration step. In some embodiments, the HIC step is performed after the viral filtration step. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0165] In some embodiments, the purification platform comprises, in order: a capture step; a conditioning step; one or more purification steps; and a UFDF 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and prior to the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0166] In some embodiments, the purification platform comprises, in order: a capture step; a conditioning step; one or more purification steps; a virus filtration step; and a UFDF 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and prior to the virus filtration step. In some embodiments, the HIC step is performed after the virus filtration step and prior to the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0167] Samples, components, and compositions obtained from the purification platform

[0168] In some aspects, the purification platforms described herein can be used to purify a target to any degree from a sample comprising the target.

[0169] 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 Escherichia coli cell.

[0170] In some embodiments, the sample has been processed, such as undergoing treatment steps prior to subjecting the sample to the purification platform described herein. In some embodiments, the sample contains a surfactant.

[0171] In some embodiments, the sample comprises 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.

[0173] In some embodiments, the target is a polypeptide. In some embodiments, the target is a polypeptide 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 moiety is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody. In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0174] 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, an esterase, a thioesterase, a phospholipase, or a ceramidase. 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.

[0175] In some cases, the purification platform described herein can comprise a plurality of purification steps. In some embodiments, the term “composition” is used herein to describe any input (except for the initial sample input to the purification platform), intermediate, or output at any stage of the purification platform. For example, in some embodiments, the use of the term “composition” is not limited to describe the final output of the purification platform.

[0176] 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.

[0177] In some embodiments, the composition comprises a target. In some embodiments, the target comprises a polypeptide. In some embodiments, the target is a polypeptide. In some embodiments, the target is a polypeptide 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 moiety is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody. In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab vedotin-piiq, certolizumab pegol, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, serpliriumab, prasinezumab, RO6874281, and RO7122290.

[0178] In some embodiments, the composition 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, an esterase, a thioesterase, a phospholipase, or a ceramidase.

[0179] Additional steps

[0180] In some aspects, the present disclosure provides additional steps involved in or associated with the purification platforms described herein. Additional steps involved in or associated with the purification platforms, and methods of performing these steps, are known. See, e.g., Liu et al. mAbs, 2, 2010, which is hereby incorporated by reference in its entirety.

[0181] In some embodiments, the purification platform further comprises a sample processing step, such as a sample preparation step. In some embodiments, the purification platform further comprises a clarification step, such as clarification of HCCF. In some embodiments, the purification platform further comprises a host cell and host cell debris removal step, such as removal of host cells and host cell debris from a sample and / or composition obtained from the purification platform. In some embodiments, the purification platform further comprises a centrifugation step. In some embodiments, the purification platform further comprises a sterile filtration step. In some embodiments, the purification platform further comprises a tangential flow microfiltration step. In some embodiments, the purification platform further comprises a flocculation / settlement step.

[0182] Methods of using the purification platform

[0183] In some aspects, the present disclosure describes methods of using the purification platforms described herein. In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform described herein.

[0184] In some aspects, provided herein is a method of reducing the rate of enzymatic hydrolysis activity of a composition obtained from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby reducing the rate of enzymatic hydrolysis activity of the composition compared to purification of the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps. In some embodiments, the relative reduction in the rate of enzymatic hydrolysis activity of the composition compared to purification of the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps is at least about 5%, such as at least about any of 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 enzyme polysorbate hydrolysis activity.

[0185] In some aspects, provided herein are methods of reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting the sample to the purification platform, thereby reducing the level of hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps. In some embodiments, the relative reduction in the level of one or more hydrolytic enzymes in the composition is at least about 5%, such as at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps. In some embodiments, the one or more hydrolytic enzymes is capable of hydrolyzing polysorbate.

[0186] In some aspects, provided herein are methods of reducing degradation of polysorbate in a composition obtained from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting the sample to the purification platform, thereby reducing degradation of polysorbate in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps. In some embodiments, the relative reduction in degradation of polysorbate in the composition is at least about 5%, such as at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps.

[0187] In some aspects, provided herein are methods of extending the shelf life of a composition obtained from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting the sample to the purification platform, thereby extending the shelf life of the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the 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 the one or more depth filtration steps and / or the one or more HIC steps is at least about 1 week, such as at least about any of 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 compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps is at least about any of 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. 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.

[0188] In some aspects, provided herein are methods of producing a composition with less degraded polysorbate from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting the sample to the purification platform, thereby producing a composition with less degraded polysorbate compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps.

[0189] In some aspects, provided herein are methods of reducing aggregation of a target in a composition obtained from a purification platform described herein comprising one or more depth filtration steps and / or one or more HIC steps, the method comprising subjecting the sample to the purification platform, thereby reducing aggregation of the target in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps and / or the one or more HIC steps.

[0190] As described herein, one or more attributes of a composition obtained from subjecting a sample to a purification platform comprising one or more depth filtration steps and / or one or more HIC steps are compared to a sample purified using the same purification platform without one or more depth filtration steps and / or one or more HIC steps. One of ordinary skill in the art will appreciate that in some instances, such a comparison must be made under appropriate conditions that allow for a meaningful comparison. For example, when comparing a composition obtained from a purification platform comprising one or more depth filtration steps and / or one or more HIC steps to a sample purified using the same purification platform without one or more depth filtration steps and / or one or more HIC steps, the temporal factor that can affect the rate of readout of enzymatic hydrolysis activity must be taken into account. Other relevant factors to take into account when comparing a composition to a reference include experimental conditions, assay used, temperature conditions, pH, timing, sample, buffer(s) used, and sample source(s).

[0191] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising a capture step; and a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture 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 comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: 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 prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to 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.

[0193] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: a capture step; a conditioning step; and one or more purification steps, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to 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.

[0194] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; and a viral filtration step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the viral filtration step. In some embodiments, the depth filtration step is performed after the viral filtration step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as a HIC step selected from a pH hold step after the one or more purification steps, one or more HIC steps after or during, such as after, the viral filtration step.

[0195] In some embodiments, the method comprises subjecting a sample comprising 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 comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the UFDF step. In some embodiments, the depth filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as one or more HIC steps selected from being performed after the one or more purification steps and / or after the UFDF step.

[0196] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; a virus filtration step; and a UFDF step, wherein the purification platform further comprises a depth filtration step. In some embodiments, the depth filtration step is performed prior to the capture step. In some embodiments, the depth filtration step is performed after the capture step and prior to the conditioning step. In some embodiments, the depth filtration step is performed after the conditioning step and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the depth filtration step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the depth filtration step is performed after the one or more purification steps and prior to the virus filtration step. In some embodiments, the depth filtration step is performed after the virus filtration step and prior to the UFDF step. In some embodiments, the depth filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second depth filtration step, such as a depth filtration step performed prior to the capture step. In some embodiments, the purification platform further comprises a HIC step, such as one or more HIC steps selected from being performed after the one or more purification steps and / or a pH hold step of the virus filtration step, after or during the virus filtration step, such as after and / or after the UFDF step.

[0197] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising 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 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 comprises subjecting a sample comprising a target to a purification platform comprising, in order: 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.

[0199] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in order: a capture step; a conditioning step; and one or more purification steps, 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0200] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: a capture step; a conditioning step; one or more purification steps; and a virus filtration 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and prior to the virus filtration step. In some embodiments, the HIC step is performed after the virus filtration step. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0201] In some embodiments, the method comprises subjecting a sample comprising 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 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 and prior to the one or more purification steps. In some embodiments, if there is more than one purification step, the HIC step is performed between any of the one or more purification steps or after the one or more purification steps. In some embodiments, the HIC step is performed after the one or more purification steps and prior to the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second HIC step, such as a HIC step performed after the one or more purification steps. In some embodiments, the purification platform further comprises a depth filtration step, such as a depth filtration step performed after the capture step.

[0202] 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; 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.

[0203] For the purposes of example and explanation of the content disclosed herein, Figure 1B The sequential workflow of options available for aspects of the exemplary purification platform 200 is illustrated below. Figure 1B As shown, 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 an EMPHAZE step performed on HCCF prior to the protein A chromatography step. TM Deep filtering step 205, XOSP deep filtering step 215 on protein A conjugate, or EMPHAZE on protein A conjugate. TM One or more deep filtering steps, any one of the deep filtering steps 220.

[0204] according to Figure 1B In some embodiments, the purification platform includes making EMPHAZE TM The deep-filtered confluent underwent EMPHAZE of HCCF prior to protein A chromatography 210. TM Deep filtration step 205. In such embodiments, the protein A complex undergoes XOSP deep filtration step 215 or EMPHAZE before downstream chromatography steps. TMDeep filtration step 220. In some embodiments, the purification platform includes an XOSP deep filtration step 215, prior to which the protein A conjugate is conditioned by adjusting the pH of the protein A conjugate to approximately 5 to approximately 6.5. In some embodiments, the purification platform includes an XOSP deep filtration step 215, wherein the XOSP deep-filtered conjugate is further subjected to HIC (such as phenyl) Rapid flow chromatography (such as) or cation exchange chromatography (such as) 50HS). In some embodiments, the purification platform includes EMPHAZE. TM Deep filtering step 220, in EMPHAZE TM Prior to deep filtration step 220, the protein A conjugate is conditioned by adjusting its pH to approximately 7 to approximately 8.5. In some embodiments, the purification platform includes EMPHAZE. TM Deep filtration step 220, EMPHAZE TM The deep-filtered merged products are further subjected to multivariate chromatography (such as Capto Adhere).

[0205] according to Figure 1B In some embodiments, the purification platform does not include EMPHAZE of HCCF. TM Deep filtration step 205. In this type of embodiment, HCCF undergoes protein A chromatography 210, and the protein A pooled product undergoes either XOSP deep filtration step 215 or EMPHAZE before downstream chromatography steps. TM Deep filtration step 220. In some embodiments, the purification platform includes an XOSP deep filtration step 215, prior to which the protein A conjugate is conditioned by adjusting the pH of the protein A conjugate to approximately 5 to approximately 6.5. In some embodiments, the purification platform includes an XOSP deep filtration step 215, wherein the XOSP deep-filtered conjugate is further subjected to HIC (such as phenyl) Rapid flow chromatography (such as) or cation exchange chromatography (such as) 50HS). In some embodiments, the purification platform includes EMPHAZE. TM Deep filtering step 220, in EMPHAZE TM Prior to deep filtration step 220, the protein A conjugate is conditioned by adjusting its pH to approximately 7 to approximately 8.5. In some embodiments, the purification platform includes EMPHAZE. TM Deep filtration step 220, EMPHAZE TM The deep-filtered merged products are further subjected to multivariate chromatography (such as Capto Adhere).

[0206] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a depth filtration step comprising processing through a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane; (b) a capture step comprising processing through protein A chromatography; and (c) a purification step, wherein the purification step comprises processing through chromatography selected from the group consisting of HIC, cation exchange chromatography, and multi-modal chromatography. In some embodiments, the depth filter is EMPHAZE TM depth filter. In some embodiments, the HIC is phenyl fast flow chromatography. In some embodiments, the cation exchange chromatography is 50HS. In some embodiments, the multi-modal chromatography is Capto Adhere.

[0207] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a first depth filtration step comprising processing through a first depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane; (b) a capture step comprising processing through protein A chromatography; (c) a second depth filtration step comprising processing through a second depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane; and (d) a purification step comprising processing through multi-modal chromatography. In some embodiments, the first depth filter and the second depth filter are EMPHAZE TM depth filter. In some embodiments, the multi-modal chromatography is Capto Adhere. In some embodiments, the second depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane is selected when the solution entering the depth filter is between about 7 and about 8.5.

[0208] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a capture step comprising processing through protein A chromatography; (b) a depth filtration step comprising processing through a depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane; and (c) a purification step comprising processing through multi-modal chromatography. In some embodiments, the depth filter is EMPHAZE TM depth filter. In some embodiments, the multi-modal chromatography is Capto Adhere. In some embodiments, the depth filter comprising a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane is selected when the solution entering the depth filter is between about 7 and about 8.5.

[0209] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a first depth filtration step comprising processing through a first depth filter; (b) a capture step comprising processing through protein A chromatography; and (c) a second depth filtration step comprising processing through a second depth filter. In some embodiments, the first depth filter comprises a hydrogel Q-functionalized nonwoven media and a multi-zone microporous membrane. In some embodiments, the first depth filter is an EMPHAZE TM depth filter. In some embodiments, the first depth filter comprises an inorganic filter aid, cellulose, and a 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 X0SP depth filter. In some embodiments, the method further comprises a conditioning step. In some embodiments, the method further comprises one or more purification steps.

[0210] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a first depth filtration step comprising processing through a first depth filter comprising a hydrogel Q- functionalized nonwoven media and a multi-zone microporous membrane; (b) a capture step comprising processing through protein A chromatography; (c) a second depth filtration step comprising processing through a second depth filter comprising silica and polyacrylic fibers; and (d) a purification step comprising processing through cation exchange chromatography. In some embodiments, the first depth filter is an EMPHAZE TM depth filter. In some embodiments, the second depth filter is an X0SP depth filter. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the second depth filter comprising silica and polyacrylic fibers is selected when the solution entering the depth filter is from about 5 to about 6.5.

[0211] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in sequence: (a) a first depth filtration step comprising processing through a first depth filter comprising a hydrogel Q- functionalized nonwoven media and a multi-zone microporous membrane; (b) a capture step comprising processing through protein A chromatography; (c) a second depth filtration step comprising processing through a second depth filter comprising silica and polyacrylic fibers; and (d) a purification step comprising processing through HIC. In some embodiments, the first depth filter is an EMPHAZE TM ​Depth filter. In some embodiments, the second depth filter is an X0SP depth filter. In some embodiments, the HIC is phenyl Fast flow chromatography. In some embodiments, the second depth filter comprising silica and polyacrylic fibers is selected when the solution entering the depth filter is about 5 to about 6.5.

[0212] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in order: (a) a capture step comprising processing by protein A chromatography; and (b) a depth filtration step comprising processing by a depth filter. In some embodiments, the depth filter comprises silica and polyacrylic fibers. In some embodiments, the depth filter is an X0SP depth filter. In some embodiments, the method further comprises an adjustment step. In some embodiments, the method further comprises one or more purification steps.

[0213] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in order: (a) a capture step comprising processing by protein A chromatography; (b) a depth filtration step comprising processing by a depth filter comprising silica and polyacrylic fibers; and (c) a purification step comprising processing by cation exchange chromatography. In some embodiments, the depth filter is an X0SP depth filter. In some embodiments, the cation exchange chromatography is Capto Adhere®. 50HS. In some embodiments, the depth filter comprising silica and polyacrylic fibers is selected when the solution entering the depth filter is about 5 to about 6.5.

[0214] In some embodiments, the method comprises subjecting a sample comprising a target to a purification platform comprising, in order: (a) a capture step comprising processing by protein A chromatography; (b) a depth filtration step comprising processing by a depth filter comprising silica and polyacrylic fibers; and (c) a purification step comprising processing by HIC. In some embodiments, the depth filter is an X0SP depth filter. In some embodiments, the HIC is phenyl Fast flow chromatography. In some embodiments, the depth filter comprising silica and polyacrylic fibers is selected when the solution entering the depth filter is about 5 to about 6.5.

[0215] Additional method steps

[0216] In some embodiments, the methods described herein further comprise additional method steps. In some embodiments, the method further comprises a cell culture step. In some embodiments, the method further comprises a formulation step, such as processing a composition to form a pharmaceutical composition or a precursor thereof.

[0217] In some embodiments, the method further comprises determining the rate of enzymatic hydrolysis activity of the composition. In some embodiments, the method further comprises performing a lipase activity assay on the composition obtained from the purification platform described herein. In some embodiments, the lipase activity assay comprises measuring the lipase activity of one or more hydrolytic enzymes by monitoring the conversion of a substrate, such as a non-fluorescent substrate, to a detectable product, such as a fluorescent product, of a hydrolytic enzyme. In some embodiments, the substrate comprises an ester bond. In some embodiments, the method further comprises determining the product of one or more hydrolytic enzymes, e.g., as described in WO2018035025, which is hereby incorporated by reference in its entirety. In some embodiments, the method further comprises determining the level of free fatty acids (FFAs) in the composition obtained from the purification platform described herein by performing a fatty acid mass spectrometry (FAMS) assay. In some embodiments, the method further comprises determining the level of one or more hydrolytic enzymes in the composition. In some embodiments, the method further comprises determining the shelf life of the composition. In some embodiments, the method further comprises determining the level of aggregates of the target in the composition.

[0218] Pharmaceutical composition

[0219] In some aspects, the present disclosure provides a pharmaceutical composition obtained from the purification platform described herein. In some embodiments, the pharmaceutical composition is obtained from the method described herein. In some embodiments, the pharmaceutical composition is a purified composition. In some embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0220] 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 a host cell impurity, such as a host cell protein, e.g., a hydrolytic enzyme.

[0221] In some embodiments, the pharmaceutical composition comprises a polysorbate. In some embodiments, the pharmaceutical composition is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0222] 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.

[0223] In some embodiments, the pharmaceutical composition has a reduced level 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] In some embodiments, the pharmaceutical composition has 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.

[0228] Formulated antibody moiety compositions

[0229] In some aspects, the present disclosure provides a formulated antibody moiety composition obtained from a purification platform described herein. In some embodiments, the formulated antibody moiety composition is obtained from a method described herein.

[0230] In some embodiments, the formulated antibody moiety composition comprises an antibody moiety. In some embodiments, the formulated antibody moiety composition comprises an antibody moiety and a polysorbate. In some embodiments, the formulated antibody moiety composition comprises an antibody moiety, a polysorbate, and a host cell impurity, such as a host cell protein, e.g., a hydrolytic enzyme.

[0231] In some embodiments, a formulated antibody moiety composition described herein has an extended shelf life 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 shelf life is assessed, such as measured, by aggregation of the antibody moiety of the formulated antibody moiety composition. In some embodiments, the shelf life is assessed, such as measured, by one or more functionalities of the antibody moiety of the formulated antibody moiety composition. In some embodiments, the shelf life is assessed, such as measured, by activity, such as binding activity, of the antibody moiety of the formulated antibody moiety composition.

[0232] In some embodiments, a formulated antibody moiety composition comprising an antibody moiety and a polysorbate has a reduced rate of polysorbate hydrolytic activity, wherein the shelf life of the composition is more than about 12 months, such as more than any of about 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months. In some embodiments, the formulated antibody moiety composition has a reduced rate of polysorbate hydrolytic activity 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 rate of polysorbate hydrolytic activity is a reduced relative rate of polysorbate hydrolytic activity.

[0233] In some embodiments, a formulated antibody moiety composition comprising an antibody moiety and a polysorbate has a reduced rate of polysorbate hydrolytic activity, wherein the shelf life of the composition is extended compared to the shelf life indicated in a document submitted to a health authority in connection with the formulated antibody moiety composition, and wherein the shelf life is extended by at least about 2 months, such as at least any of 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 indicated in the document. In some embodiments, the formulated antibody moiety composition has a reduced rate of polysorbate hydrolytic activity 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 rate of polysorbate hydrolytic activity is a reduced relative rate of polysorbate hydrolytic activity.

[0234] In some embodiments, a formulated antibody moiety composition comprising an antibody moiety and a polysorbate has reduced polysorbate degradation compared to the degradation indicated in a document submitted to a health authority in connection with the formulated antibody moiety composition, wherein the degradation is reduced by at least about 5%, such as at least any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the formulated antibody moiety composition has reduced polysorbate degradation 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 degradation is a reduced relative polysorbate degradation.

[0235] In some embodiments, the rate of polysorbate hydrolysis activity of the formulated antibody moiety composition is reduced by at least about 5%, such as at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a reference.

[0236] In some embodiments, the formulated antibody moiety composition comprises an antibody moiety and a polysorbate, wherein the polysorbate degrades about 50% or less per year, such as about any of 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 during storage of the liquid composition.

[0237] In some embodiments, the reduced aggregate formation of the formulated antibody moiety compositions described herein is for at least about 6 months, such as at least about any of 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, 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, at least about 20% less (such as at least about any of 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) aggregate formation of the formulated antibody moiety compositions described herein is for at least about 6 months, such as at least about any of 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 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. Methods for assessing, e.g., measuring, aggregate formation are known in the art and include, e.g., visual inspection, dynamic light scattering, static light scattering, and optical density measurements.

[0238] In some embodiments, the formulated antibody moiety composition described herein retains at least about 50% (such as at least about any of 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) of the antibody moiety activity compared to a reference for at least about 6 months, such as at least about any of 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, wherein the reference is 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.

[0239] In some embodiments, the antibody moiety is a monoclonal antibody.

[0240] In some embodiments, the antibody moiety is a human antibody, a humanized antibody, or a chimeric antibody.

[0241] In some embodiments, the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0242] In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0243] In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0244] Other aspects reported herein are formulated antibody compositions that have low sorbitol ester degradation during storage. One aspect of the invention is a formulated antibody composition comprising an antibody / protein and a polysorbate ester, wherein the polysorbate ester degrades 20% or less per year (in one embodiment 15% or less, in one embodiment 12% or less, in one embodiment 10% or less, in one embodiment 9% or less, in one embodiment 8% or less, in one embodiment 7% or less, in one embodiment 6% or less, in one embodiment 5% or less, in one embodiment 4% or less, in one embodiment 3% or less, in one embodiment 2% or less, in one embodiment 1% or less) during the storage / shelf life of the formulated antibody composition. In one embodiment, the polysorbate ester degrades 10% or less per year during storage of the liquid composition.

[0245] Another aspect is a formulated antibody composition comprising an antibody and a polysorbate ester, wherein the polysorbate ester is present in the composition at a concentration of at least 80% (in one embodiment 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%) of the initial concentration after one year, wherein the initial concentration is the concentration at which the antibody is formulated or initially stored in the liquid composition.

[0246] Exemplary embodiments

[0247] Embodiment 1. A method of reducing the rate of enzymatic hydrolytic activity 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 hydrolytic activity of the composition compared to purifying the sample using the same purification platform without the depth filtration step.

[0248] Embodiment 2. The method of embodiment 1, wherein the rate of enzymatic hydrolytic activity is the rate of enzymatic polysorbate hydrolytic activity.

[0249] Embodiment 3. The method of embodiment 1 or 2, wherein the relative reduction in the rate of enzymatic hydrolytic activity of the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0250] Example 4. A method of 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 depth filtration step, thereby reducing the level of hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the depth filtration step.

[0251] Example 5. The method of Example 4, wherein the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate esters.

[0252] Example 6. The method of 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.

[0253] Example 7. A method for reducing 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 comprising: (a) a capture step; and (b) a depth filtration step, thereby reducing degradation of polysorbate esters in the composition compared to purifying the sample using the same purification platform without the depth filtration step.

[0254] Example 8. The method of Example 7, wherein the relative reduction in degradation of the 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.

[0255] Example 9. The method of any one of Examples 1-8, wherein the purification platform is used to purify a target from the sample, wherein the sample comprises the target and one or more host cell impurities.

[0256] Example 10. The method of Example 9, wherein the target comprises a polypeptide.

[0257] Example 11. The method of Example 9 or 10, wherein the host cell impurity is a host cell protein.

[0258] Example 12. The method of any one of Examples 1-11, wherein the depth filtration step is performed prior to the capture step, or the depth filtration step is performed after the capture step.

[0259] Example 13. The method of any one of Examples 1-12, wherein the depth filtration step comprises processing through a depth filter.

[0260] Example 14. The method of Example 13, wherein the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, a cellulose fiber, a polymer fiber, a tacky resin, and an ash composition.

[0261] Example 15. The method of Example 14, wherein at least a portion of the substrate of the depth filter comprises a surface modification.

[0262] Example 16. The method of Example 15, wherein the surface modification is one or more of a quaternary amine surface modification, a cationic surface modification, and an anionic surface modification.

[0263] Example 17. The method of any one of Examples 14-16, wherein the depth filter is selected from the group consisting of EMPHAZE® TM EMPHAZE® PDD1 depth filter, ZETA PLUS TM 120ZA depth filter, and ZETA PLUS TM 120ZB depth filter.

[0264] Example 18. The method of any one of Examples 1-17, wherein the capturing step comprises processing by affinity chromatography.

[0265] Example 19. The method of 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, kappa chromatography, and kappa XL chromatography.

[0266] Example 20. The method of any one of Examples 1-19, wherein the purification platform further comprises a viral inactivation step, wherein the viral inactivation step is performed after the capturing step.

[0267] Example 21. The method of Example 20, wherein the depth filtration step is performed after the viral inactivation step.

[0268] Example 22. The method of any one of Examples 1-21, wherein the purification platform further comprises another depth filtration step performed prior to the capturing step.

[0269] Example 23. The method of 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 capturing step, the depth filtration step, and the viral inactivation step, if present.

[0270] Example 24. The method of Example 23, wherein the one or more purification steps comprise a polypeptide purification step.

[0271] Example 25. The method of Example 23 or 24, wherein the purification platform further comprises another depth filtration step performed before, between, or after the one or more purification steps.

[0272] Example 26. The method of any one of Examples 1-25, wherein the purification platform further comprises an ultrafiltration / diafiltration (UFDF) step, and wherein the UFDF step is performed after the one or more purification steps.

[0273] Example 27. The method of Example 26, wherein the purification platform further comprises another depth filtration step performed before or after the UFDF step.

[0274] Example 28. The method of any one of Examples 1-27, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step.

[0275] Example 29. The method of Example 28, wherein the HIC purification step is performed before, between, or after the one or more purification steps, if present.

[0276] Example 30. The method of Example 28, wherein the HIC purification step is performed after the one or more purification steps and before the UFDF step, if present.

[0277] Example 31. The method of Example 26 or 27, wherein the purification platform further comprises a pH hold step, wherein the pH hold step is performed after the one or more purification steps, if present, and before the UFDF step.

[0278] Example 32. The method of Example 31, wherein the purification platform further comprises a viral filtration step, wherein the viral filtration step is performed after the pH hold step and before the UFDF step.

[0279] Example 33. The method of Example 32, wherein the viral filtration step comprises processing through a viral filter.

[0280] Example 34. The method of Example 28, wherein the HIC purification step comprises processing through a HIC filter.

[0281] Embodiment 35. The method of any one of embodiments 23-34, wherein the one or more purification steps each independently comprise processing by chromatography selected from the group consisting of: ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induction chromatography, ceramic hydroxyapatite chromatography, and multimodal chromatography.

[0282] Embodiment 36. The method of any one of embodiments 23-35, wherein the one or more purification steps each independently comprise processing by chromatography selected from the group consisting of: DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel TM , Capto MMC, and Capto Adhere.

[0283] Embodiment 37. A method of 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, the purification platform comprising, in the following order: (a) a capture step comprising processing by affinity chromatography; (b) a viral inactivation step; (c) a second polypeptide purification step; (d) a third polypeptide purification step; and (e) an ultrafiltration / diafiltration (UFDF) step, wherein the purification platform further comprises a depth filtration step performed upon one or more of: (i) prior to the capture step; (ii) after the capture step and prior to the viral inactivation step; (iii) after the viral inactivation step and prior to the second polypeptide purification step; (iv) after the second polypeptide purification step and prior to the third polypeptide purification step; or (v) after the third polypeptide purification step and prior to the ultrafiltration / diafiltration (UFDF) step; thereby reducing the rate of enzymatic hydrolysis activity of the composition compared to purification of the sample using the same purification platform without the depth filtration step.

[0284] Embodiment 38. The method of embodiment 37, wherein the purification platform further comprises, in the following order, a pH hold step and a viral filtration step performed after the third polypeptide purification step and prior to the UFDF step.

[0285] Embodiment 39. The method of embodiment 38, wherein the viral filtration step comprises processing by a viral filter.

[0286] Embodiment 40. The method of any one of embodiments 37-39, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step performed upon one or more of: (i) after the third polypeptide 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.

[0287] Embodiment 41. The method of any one of embodiments 1-40, further comprising determining the rate of enzymatic hydrolysis activity of the composition.

[0288] Embodiment 42. The method of any one of embodiments 1-41, further comprising determining the level of one or more hydrolytic enzymes in the composition.

[0289] Embodiment 43. The method of any one of embodiments 1-42, wherein the composition comprises a polysorbate.

[0290] Embodiment 44. The method of embodiment 43, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0291] Embodiment 45. The method of any one of embodiments 1-44, further comprising a sample processing step.

[0292] Embodiment 46. The method of any one of embodiments 1-45, wherein the sample is or is derived from a cell culture sample.

[0293] Embodiment 47. The method of embodiment 46, wherein the cell culture sample comprises a host cell, and wherein the host cell is a Chinese hamster ovary (CHO) cell or an Escherichia coli cell.

[0294] Embodiment 48. The method of any one of embodiments 1-47, wherein the sample comprises a host cell or a component derived from the host cell.

[0295] Embodiment 49. The method of any one of embodiments 1-48, wherein the sample comprises one or more host cell proteins, and wherein one of the one or more host cell proteins is a hydrolytic enzyme.

[0296] Embodiment 50. The method of embodiment 49, wherein the hydrolytic enzyme is a lipase, an esterase, a thioesterase, a phospholipase, or a ceramidase.

[0297] Example 51. The method of any one of Examples 1-50, wherein the sample comprises a target, and wherein the target is an antibody moiety.

[0298] Example 52. The method of Example 51, wherein the antibody moiety is a monoclonal antibody.

[0299] Example 53. The method of Example 51 or 52, wherein the antibody moiety is a human antibody, a humanized antibody, or a chimeric antibody.

[0300] Example 54. The method of any one of Examples 51-53, wherein the antibody moiety is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0301] Example 55. The method of any one of Examples 51-54, wherein the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0302] Example 56. A pharmaceutical composition obtained by the method of any one of Examples 1-55.

[0303] Example 57. A formulated antibody moiety composition comprising an antibody moiety and a polysorbate, wherein the composition has a reduced rate of polysorbate hydrolysis activity, wherein the shelf life of the composition exceeds 24 months.

[0304] Example 58. A formulated antibody moiety composition comprising an antibody moiety and a polysorbate, wherein the composition has a reduced rate of polysorbate hydrolysis activity, wherein the shelf life of the composition is extended compared to the shelf life indicated in documents submitted to health authorities in connection with the formulated antibody moiety composition, wherein the shelf life is extended by at least 6 months compared to the shelf life indicated in the documents.

[0305] Example 59. A formulated antibody moiety composition comprising an antibody moiety, wherein the formulated antibody moiety composition has reduced polysorbate degradation, wherein the degradation is reduced by at least about 20% compared to the degradation indicated in documents submitted to health authorities in connection with the formulated antibody moiety composition.

[0306] Example 60. A formulated antibody moiety composition comprising an antibody moiety and a polysorbate, wherein the polysorbate degrades 20% or less per year during storage of the liquid composition.

[0307] Example 61. The formulated antibody moiety composition of any one of Examples 57-60, wherein the antibody moiety is a monoclonal antibody.

[0308] Example 62. The formulated antibody moiety composition of any one of Examples 57-61, wherein the antibody moiety is a human antibody, a humanized antibody, or a chimeric antibody.

[0309] Example 63. The formulated antibody moiety composition of any one of Examples 57-62, wherein the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0310] Example 64. The formulated antibody moiety composition of any one of embodiments 57-63, wherein the antibody moiety is selected from the group consisting of: ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, omalizumab, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0311] Example 65. The formulated antibody moiety composition of any one of embodiments 57-64, wherein the polysorbate hydrolytic activity rate is reduced by at least about 20%.

[0312] Example 66. The formulated antibody moiety composition of any one of embodiments 57-65, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0313] Example 67. A method of reducing an enzymatic hydrolytic activity rate of a composition obtained from a purification platform, the method comprising subjecting a sample to the purification platform, the purification platform comprising, in order: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step; or after the capture step and prior to the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the enzymatic hydrolytic activity rate of the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

[0314] Example 68. The method of embodiment 67, wherein the enzymatic hydrolytic activity rate is an enzymatic polysorbate hydrolytic activity rate.

[0315] Example 69. The method of embodiment 67 or 68, wherein the relative reduction in the enzymatic hydrolytic activity rate of the composition is at least about 20% compared to purifying the sample using the same purification platform without the depth filtration step.

[0316] Example 70. A method of 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 order: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step and prior to the purification step; or after the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membranes; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing 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.

[0317] Example 71. The method of Example 70, wherein the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate esters.

[0318] Example 72. The method of 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.

[0319] Example 73. A method of reducing 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 order: (a) a capture step comprising processing by affinity chromatography; and (b) a purification step comprising processing by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more depth filtration steps, wherein the one or more depth filtration steps are performed: prior to the capture step; after the capture step; or after the capture step and prior to the purification step, wherein each depth filtration step comprises processing by a depth filter, and wherein the depth filter comprises a material selected from the group consisting of: (i) silica and polyacrylic fibers; (ii) hydrogel Q (quaternary amine)-functionalized nonwoven media and multi-zone microporous membrane; and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing degradation of polysorbate in the composition compared to purifying the sample using the same purification platform without the one or more depth filtration steps.

[0320] Example 74. The method of 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.

[0321] Example 75. The method of any one of Examples 67-74, wherein the depth filter comprising the silica and the polyacrylic fibers comprises silica filter aid and polyacrylic fiber pulp.

[0322] Example 76. The method of any one of Examples 67-74, wherein the depth filter comprising the hydrogel Q-functionalized nonwoven media and the multi-zone microporous membrane comprises four layers comprising hydrogel Q-functionalized nonwoven material and nine-zone microporous membrane.

[0323] Example 77. The method of 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 resinous binder.

[0324] Example 78. The method of any one of Examples 67-77, wherein the depth filter is selected based on the pH of a solution entering the depth filter.

[0325] Example 79. The method of Example 78, wherein the depth filter comprising the silica and the polyacrylic fiber is selected when the solution entering the depth filter is from about 5 to about 6.5.

[0326] Example 80. The method of Example 78, wherein the depth filter comprising the hydrogel Q-functionalized nonwoven media and the multi-zone microporous membrane is selected when the solution entering the depth filter is from about 7 to about 8.5.

[0327] Example 81. The method of any one of Examples 67-80, further comprising selecting the depth filter based on the pH of the solution entering the depth filter.

[0328] Example 82. The method of any one of Examples 67-81, wherein the purification platform comprises, in sequence: a depth filtration step comprising processing by the depth filter comprising the hydrogel Q-functionalized nonwoven media and multi-zone microporous membrane; the capture step comprising processing by protein A chromatography; and the purification step.

[0329] Example 83. The method of Example 82, wherein the purification step comprises processing by the HIC.

[0330] Example 84. The method of Example 83, wherein the HIC is phenyl fast flow chromatography.

[0331] Example 85. The method of Example 82, wherein the purification step comprises processing by the cation exchange chromatography.

[0332] Example 86. The method of Example 85, wherein the cation exchange chromatography is 50HS.

[0333] Example 87. The method of any one of Examples 67-86, wherein the purification platform further comprises a second depth filtration step comprising processing by the depth filter comprising the silica and the polyacrylic fiber, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0334] Example 88. The method of Example 82, wherein the purification step comprises processing by the multi-modal chromatography.

[0335] Example 89. The method of Example 88, wherein the multi-modal chromatography is Capto Adhere.

[0336] Example 90. The method of Example 88 or 89, wherein the purification platform further comprises a second depth filtration step comprising processing through the depth filter comprising the hydrogel Q-functionalized nonwoven media and the multi-zone microporous membrane, and wherein the second depth filtration step occurs after the capture step and before the purification step.

[0337] Example 91. The method of any one of Examples 67-90, wherein the purification platform is used to purify a target from the sample, wherein the sample comprises the target and one or more host cell impurities.

[0338] Example 92. The method of Example 91, wherein the target comprises a polypeptide.

[0339] Example 93. The method of Example 91 or 92, wherein the host cell impurity is a host cell protein.

[0340] Example 94. The method of any one of Examples 67-93, wherein the purification platform further comprises a viral inactivation step, wherein the viral inactivation step occurs after the capture step.

[0341] Example 95. The method of Example 94, wherein the one or more depth filtration steps occur after the viral inactivation step.

[0342] Example 96. The method of any one of Examples 67-95, wherein the purification platform further comprises an ultrafiltration / diafiltration (UFDF) step, and wherein the UFDF step occurs after the purification step.

[0343] Example 97. The method of any one of Examples 67-96, further comprising determining the rate of enzymatic hydrolysis activity of the composition.

[0344] Example 98. The method of any one of Examples 67-97, further comprising determining the level of one or more hydrolytic enzymes in the composition.

[0345] Example 99. The method of any one of Examples 67-98, wherein the composition comprises a polysorbate.

[0346] Example 100. The method of Example 99, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0347] Example 101. The method of any one of Examples 67-100, further comprising a sample processing step.

[0348] Example 102. The method of any one of Examples 67-101, wherein the sample is or is derived from a cell culture sample.

[0349] Example 103. The method of 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.

[0350] Example 104. The method of any one of Examples 67-103, wherein the sample comprises or is derived from components of host cells.

[0351] Example 105. The method of any one of Examples 67-104, wherein the sample comprises one or more host cell proteins, and wherein one of the one or more host cell proteins is a hydrolase.

[0352] Example 106. The method of Example 105, wherein the hydrolase is a lipase, an esterase, a thioesterase, a phospholipase, or a ceramidase.

[0353] Example 107. The method of any one of Examples 67-106, wherein the sample comprises a target, and wherein the target is an antibody moiety.

[0354] Example 108. The method of Example 107, wherein the antibody moiety is a monoclonal antibody.

[0355] Example 109. The method of Example 107 or 108, wherein the antibody moiety is a human antibody, a humanized antibody, or a chimeric antibody.

[0356] Example 110. The method of any one of examples 107-109, wherein the antibody moiety is selected from the group consisting of an anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-IgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-L1 antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-Factor D antibody, an anti-Factor IX antibody, an anti-Factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, and a TYRP1 TCB antibody.

[0357] Example 111. The method of any one of examples 107-110, wherein the antibody moiety is selected from the group consisting of: ocrelizumab, pertuzumab, trastuzumab, tocilizumab, farletuzumab, polatuzumab, gantenerumab, certolizumab, crizanlizumab, mosunetuzumab, tiraguliximab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lekinumab, olamkicept, ranibizumab, emicizumab, seruplinumab, prasinezumab, RO6874281, and RO7122290.

[0358] Example 112. A pharmaceutical composition obtained by the method of any one of examples 67-111.

[0359] Those skilled in the art will recognize, within the scope and spirit of the disclosure herein, that several embodiments are possible. The disclosure is further illustrated by the following examples, which should not be construed as limiting the disclosure in scope or in spirit to the specific procedures described herein.

[0360] Example

[0361] Example 1

[0362] This example demonstrates a comparison between two purification platforms for purifying the antibody trastuzumab from an unconditioned bulk using: (1) a common purification platform; and (2) the same purification platform including an additional PDD1 depth filtration step after conditioning the eluate from affinity chromatography and prior to cation exchange (CEX) chromatography.

[0363] The common purification platform (1) was performed twice and consisted of the following sequential steps: affinity chromatography, eluate conditioning, cation exchange chromatography, anion exchange chromatography, and tangential flow filtration and conditioning of the resulting pool from anion exchange chromatography. The pool names and pool descriptions for the purification process are shown in Table 1 below.

[0364] Table 1. Purification pool names and pool descriptions.

[0365] Pool Name Pool Description Adjusted affinity pool Affinity pool with adjusted pH Deep bed filtration pool Filtration pool (flow through) Anion exchange pool Anion exchange chromatography elution pool Adjusted anion exchange pool Anion exchange pool with adjusted pH Unadjusted bulk Tangential flow filtration pool adjusted with diafiltration buffer

[0366] The purification platform with the addition of a PDD1 depth filtration step (2) was performed twice. The polysorbate hydrolysis activity at unconditioned bulk level was compared by free fatty acid mass spectrometry (FAMS), the methodology of which is disclosed in more detail in the Materials and Methods section.

[0367] For the purification platform including a depth filtration step, the eluate was conditioned after affinity chromatography and prior to filtration through a PDD1 depth filter (Pall PDD1; SUPRAcap TM - 50 SC050 PDD1 (Lot: 102992583); Area: 22 cm 2 ) filter. The PDD1 depth filter was equilibrated using CEX equilibration buffer. Filtration of the conditioned affinity pool was pressure controlled. The filtration step was performed at room temperature (15-30 °C). Trastuzumab flowed through the PDD1 filter. The PDD1 depth filter was rinsed with CEX equilibration buffer prior to use and after filtration. The PDD1 depth filter was discarded after each use. The acceptable range for the CEX equilibration buffer was: 0.020-0.040 M MES (2-(N-morpholino)ethanesulfonic acid), 0.042-0.048 M NaCl, pH 5.50-5.70, and a conductivity of 5.10-5.70 mS / cm. The operating conditions for the PDD1 depth filter are shown in Table 2 below.

[0368] Table 2. PDD1 depth filter operating conditions.

[0369]

[0370] Cation exchange chromatography (SP FF chromatography) was performed in bind-and-elute 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 step elution. All chromatography steps were performed at ambient temperature (15-30 °C).

[0371] The affinity pool is adjusted by adjusting the pH to 5.5 ± 0.3 with Tris base prior to loading onto the cation exchange column. If the pool is over-titrated, the pool is adjusted to the specified pH with citric acid and then the conductivity is adjusted to 3.5 ± 1.0 mS / cm with the addition of high purity water, if necessary. The cation exchange column is equilibrated with equilibration buffer and loaded with the adjusted affinity pool. After loading, the column is washed with equilibration buffer and then washed with a gradient of increasing conductivity followed by a wash with equilibration buffer again. The trastuzumab is eluted from the column by step elution with elution buffer. Collection of eluate is started and stopped based on absorbance and volume.

[0372] The acceptable range for the CEX equilibration buffer is: 0.020-0.040 M MES, 0.042-0.048 M NaCl, pH 5.50-5.70, and a conductivity of 5.10-5.70 mS / cm. The acceptable range for the elution buffer is: 0.020-0.040 M MES, 0.092-0.098 M NaCl, pH 5.50-5.70, and a conductivity of 10.10-10.80 mS / cm. The operating conditions for the cation exchange chromatography are shown in Table 3 below.

[0373] Table 3. Cation exchange chromatography operating conditions.

[0374]

[0375]

[0376] a grams of trastuzumab per liter of SP Cation exchange resin.

[0377] Anion exchange chromatography (Q chromatography) is performed in flow-through mode and reduces CHO HCP, DNA, Protein A, and potential viruses. Trastuzumab flows through the column under the loading and wash conditions employed. All chromatography steps are performed at ambient temperature (15-30 °C).

[0378] The pH of the cation exchange pool is adjusted to pH 8.0 ± 0.5 with Tris base and MES, if necessary, and the conductivity is adjusted to 5.5-7.8 mS / cm with high purity water. The anion exchange column is equilibrated with equilibration buffer and then loaded with the pH adjusted cation exchange pool. After loading is complete, the column is washed with equilibration buffer. The pool is combined based on absorbance and volume. The pH of the anion exchange pool is adjusted to 6.0 ± 0.1 with acetic acid.

[0379] 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.

[0380] Table 4. Operating conditions for anion exchange chromatography.

[0381]

[0382] 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 polyether sulfone (PES) membrane. Subsequently, the buffer composition was adjusted to meet the conditions by adding a solution containing histidine.

[0383] 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.

[0384] 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.

[0385] Table 5. TFF Operating Conditions.

[0386]

[0387] 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 reduction in host cell protein levels compared to a conventional purification platform.

[0388] Table 6. Host cell proteins measured in cation exchange chromatography loading compositions.

[0389]

[0390] 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 a decrease in host cell protein levels compared to a conventional purification platform.

[0391] Table 7. Host cell proteins of TFF conjugates after conditioning with percolation composition.

[0392]

[0393]

[0394] The hydrolytic activity in the TFF confluence after dialysis conditioning was measured, and the results are provided 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 using 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 ).

[0395] Materials and Methods

[0396] Protein concentration determination. Using ultraviolet spectroscopy. 50 UV-Vis spectrophotometer (Varian) or NanoDrop TM OneC (Thermo Scientific) determines protein concentration. Protein samples are diluted in their respective buffers and measured twice. Concentration is determined according to the following equation derived from Lambert-Beer's law: = (280nm–320nm) / ··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 are 1.48, 1.7, and 1.35 ml / (mg·cm), respectively.

[0397] Lipase activity assay (LEAP assay). Lipase activity assay measures lipase activity by monitoring the conversion of a non-fluorescent substrate (4-MU, ChemImpex Int'l Inc) to a fluorescent product (MU, Sigma-Aldrich) through cleavage of the substrate ester bond. Protein pool samples to be analyzed were re-buffered to 150 mM Tris-Cl pH 8.0 by using Amicon Ultra-0.5 ml centrifugal filter units (10,000 Da cut-off, Merck Millipore). The assay reaction mixture contained 80 μL 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 4-MU substrate (1 mM in DMSO) and 10 μL protein pool sample. Protein pool sample concentration was adjusted to 10-30 g / L and tested at three different concentrations. Each reaction was performed in technical triplicate in a 96-well half-area polystyrene plate (black with lid and transparent flat bottom, Corning Incorporated) and the increase in fluorescence signal (excitation at 355 nm, emission at 460 nm) was monitored every 10 minutes by incubating the reaction plate at 37 °C for two hours in an Infinite 200 Pro plate reader (Tecan Life Sciences). The rate of MU production was derived from the slope of the fluorescence time course (0.5-2 hours) and expressed as the raw rate (k raw [RFU / h]).

[0398] In addition, an enzyme blank reaction was established to measure any non-enzymatic cleavage of the substrate caused by the buffer matrix. 10 μL protein pool sample was replaced by 10 μL 150 mM Tris-Cl pH 8.0 in the reaction mixture. The rate of self-cleavage (k self [RFU / h]) was derived from the slope of the fluorescence time course (0.5-2 hours). To convert the fluorescence signal (RFU) to μΜ of MU, a standard MU was added three times to each plate. To 10 μL MU (100 μΜ in DMSO) was supplemented with 10 μL 150 mM Tris-Cl pH 8.0 and 80 μL reaction buffer. The conversion factor a [RFU / μΜ] was calculated by averaging the fluorescence signal (0.5-2 hours) and dividing it by the final concentration of MU present in the well.

[0399] The lipase activity of a sample given in [μΜ MU / h] was calculated by subtracting the reaction rate of the enzyme blank (k raw [RFU / h]) from the reaction rate of the sample (k self- cleavage [RFU / h]) and converted to pmol MU / h by dividing this term by the conversion factor a [RFU / pmM]. The activity was normalized to the protein concentration applied per well. To report the hydrolytic activity in percent, the lipase activity of the reference sample was set to 100%.

[0400] Free fatty acid and mass spectrometry (FAMS) assay. To monitor the content of free fatty acids after PS20 degradation in each elution fraction, samples were first prepared for PS20 stability studies and subsequently analyzed by mass spectrometry. Protein pool samples were adjusted to the same protein concentration (as indicated in the respective experimental instructions) containing 0.04% (w / v) SR-PS20, 10 mM L-methionine and 100 mM Tris pH 8, unless otherwise stated. L-methionine was added as an effective antioxidant to control the oxidative degradation of PS20 during the experimental time course. As buffer control samples, the applied protein volume was replaced by the same volume of the respective elution buffer system.

[0401] All reaction mixtures were incubated in a Thermomixer (Eppendorf) at 37°C or 40°C with shaking at 600 rpm. Samples were taken after defined time points (as indicated in the respective figures) and stored at -80°C until subsequent analysis.

[0402] Fifty pL of sample were transferred into a new Eppendorf cup. Two hundred pL of FFA solvent solution (500 ng / mL D 23 - lauric acid and 500 ng / mL 13 C 14 - myristic acid in acetonitrile) were added and vortexed briefly. Samples were centrifuged at 14.000 rpm for 5 min and transferred into HPLC vials for MS analysis. On a Thermo Scientific TM Vanquish TM UHPLC system using ACQUITY Peptide BEH C18 chromatography column (1.7 pm 2.1 x 150 mm and ) from 5 pL injection samples. Eluent A (0.1% ammonium hydroxide in water) and eluent B (100% acetonitrile) were used for the following gradient with 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, eluent A was increased to 100% and held until 6.0. Eluent B was set to 70% at 6.1 min and held until 10.0 min to reach equilibrium. The mass spectrometer (Triple The 6600 (AB Sciex) operates in negative ionization mode with an ion spray voltage of –4500V. The source temperature is set at 450℃, and the TOF mass range is 100-1000 m / Z. The declustering potential is -120V, and the collision energy is -10V.

[0403] Large amounts of lauric acid, myristic acid, and isotopically labeled (D) were generated. 23 )-Lauric acid and ( 13 C 14 XIC of myristic acid. Integrating each peak and determining 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%.

[0404] Example 2

[0405] 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 depth filtration step performed after conditioning the eluent from affinity chromatography and before cation exchange (CEX) chromatography; and (3) a platform with an added EMPHAZE step performed after conditioning the eluent from affinity chromatography and before cation exchange (CEX) chromatography. TM A standard purification platform for deep filtration steps.

[0406] 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.

[0407] For purification platforms that include a depth filtration step, the eluent is conditioned after affinity chromatography and before filtration through the depth filter. The PDD1 depth filter used is Pall PDD1, SUPRAcap. TM -50SC050PDD1 (Batch No.: 102992583), Area: 22cm² 2 The EMPHAZE used TM The depth filter is EMPHAZE TM AEX Hybrid (batch number: S210650302), area: 25cm² 2 Trastuzumab was passed through a depth filter. The filtration process was performed at ambient temperature (15℃-30℃).

[0408] Prior to use, the depth filter is equilibrated with the cation exchange equilibration buffer. The filtration of the adjusted affinity pool is flow rate controlled. No rinse is performed after filtration. This allows for the actual reduction of host cell proteins (enzymes) to be checked. The filter is discarded after each use. The acceptable range for the CEX equilibration buffer is: 0.020-0.040 M MES, 0.042-0.048 M NaCl, pH 5.50-5.70, and a conductivity of 5.10-5.70 mS / cm. The operating conditions for the depth filter are shown in Table 8 below.

[0409] Table 8. Depth filter operating conditions.

[0410]

[0411] The amount of host cell proteins in the cation exchange chromatography load composition was measured and the results are provided in Table 9. Protein concentration measurements were performed according to Example 1. Compared to the regular purification platform, both purification platforms with the EMPHAZE TM A reduction in host cell protein levels was observed for both the purification platform with the depth filter and the purification platform with the PDD1 depth filter.

[0412] Table 9. Measured host cell proteins in the cation exchange chromatography load composition.

[0413]

[0414] The lipase activity of the cation exchange chromatography load of the regular platform (after affinity chromatography, no depth filtration, reference) was compared to the corresponding cation exchange chromatography load including an additional depth filtration step after affinity chromatography Figure 3 ) according to Example 1.

[0415] The hydrolytic activity of the cation exchange chromatography load of the regular platform (after affinity chromatography, no depth filtration, reference) was compared to the corresponding cation exchange chromatography load including an additional depth filtration step after affinity chromatography Figure 4 ) according to Example 1. The 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. Both purification platforms including a depth filter observed a reduction in the rate of enzyme hydrolytic activity as measured by the lipase activity assay Figure 3 ) and a reduction in the amount of FAA in the FAMS assay Figure 4

[0416] Example 3

[0417] ​This example demonstrates the purification of an anti-VEGF / Ang2 antibody using the purification platform, using a depth filtration step performed on the HCCF (harvested cell fluid) prior to sterilization and a second depth filtration step performed after affinity chromatography (CaptureSelect TM FcXL). The purification platform used is described in detail in Figure 5 . The reference control was performed using the purification process shown, without the additional HCCF depth filtration step Figure 5 .

[0418] As shown in Figure 5 , the HCCF was filtered through three different depth filters designed to remove potential host cell proteins. The antibody was flowed through the filters. The filtration steps were performed at ambient temperature (15-30°C).

[0419] Prior to use, the three depth filters were equilibrated using the affinity equilibration buffer for EMPHAZE TM (EMPHAZE TM AEX Hybrid (batch number: S228585702), area: 25 cm 2 ), VR02 (BioCap_VR02 (batch number: 3923452), area: 25 cm 2 ) and 120ZB (BioCap_120ZB (batch number: 3923452), area: 25 cm 2 ) and the cation exchange equilibration buffer for PDD1 (SUPRAcap TM -50SC050 PDD1 (batch number: 103119429), area: 22 cm 2 ). The filtration of the HCCF was pressure controlled (feed pressure = 0.2 MPa; maximum feed flow rate: 25 mL / min). The filtration of the conditioned affinity pool was flow rate controlled (feed flow rate = 5.2 ml / min; pressure control: 0.2 MPa). After filtration, the filters were rinsed with the same buffer to recover the product. The filters were discarded after each use. The equilibration buffers are provided in Table 10.

[0420] Table 10. Depth filter equilibration buffers.

[0421] Filter Equilibration buffer Filter 1 (EMPHAZE TM )]]> 25 mM Tris, 25 mM NaCl, pH 7.20 Filter 2 (VR02) 25 mM Tris, 25 mM NaCl, pH 7.20 Filter 3 (120ZB) 25 mM Tris, 25 mM NaCl, pH 7.20 Filter 4 (PDD1) 50 mM Tris / Acetate, pH 7.20

[0422] PDD1 filter rinsing was performed using 200 mL (~ 90 L / m 2 ) of water for injection. EMPHAZE TM filter rinsing was performed using 200 mL (~ 90 L / m 2 ) of CI equilibration buffer. The volume of HCCF filtered was 1750 mL (for EMPHAZETM ~700L / m 2 ).

[0423] The operating conditions for CaptureSelect FcXL are shown in Table 11 below.

[0424] Table 11. Operation conditions for CaptureSelect FcXL.

[0425]

[0426] The amount of host cell protein loaded was measured at different points on the purification platform (see [reference]). Figure 5 The results are provided in Table 12.

[0427] Table 12. Measured host cell proteins in the compositions obtained on the purification platform. Asterisks indicate... Figure 5 The sampling points are shown.

[0428]

[0429] 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 (VR02 and 120ZB) It was observed that the enzymatic hydrolysis rate of FcXL eluent decreased under different purification platforms. Figure 6A Compared to a standard purification platform without an attached depth filter, using any test depth filter (EMPHAZE) TM (VR02 and 120ZB) It was observed that the enzymatic hydrolysis activity rate of PDD1 filtrate decreased under different purification platforms. Figure 6B ).

[0430] FAMS assays were performed to compare two purification platforms used for purifying antibody pools from strong cation exchange chromatography: (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 using the 120 ZB deep filter showed a decrease in the rate of enzymatic hydrolysis activity, as measured by the amount of free fatty acids. Figure 7 ).

[0431] Example 4

[0432] This example demonstrates a comparison of purification platforms for purifying anti-FAP-IL2v that incorporate two different depth filters, X0SP or PDD1, for filtering the conditioned affinity chromatography (protein A chromatography) eluate.

[0433] HCCF samples were prepared and filtered as described in Example 1.

[0434] Lipase activity of protein A chromatography eluate of the common platform (without depth filtration) was compared to affinity chromatography eluate of purification platforms that included depth filtration, where the protein A chromatography eluate was subjected to an X0SP or PDD1 depth filtration step. Lipase activity assays were performed according to Example 1. Results of lipase activity in fractions obtained from the X0SP depth filter are shown in Figure 8A . Results of lipase activity obtained from the PDD1 depth filter are shown in Figure 8B .

[0435] Example 5

[0436] This example demonstrates purification optimization experiments for purifying various antibody moieties that were performed to find selections that minimize polysorbate hydrolytic degradation in the antibody moiety composition obtained from the purification platform. The experiments disclosed herein evaluate the inclusion of depth filters such as EMPHAZE TM as a protein A and second chromatography column load filter, and HIC media (ZORBAX phenyl membrane) as a polish column eluate pool filter or load filter for a subsequent viral filtration step.

[0437] Several depth filters, including EMPHAZE TM and X0SP, were evaluated for their potential to remove or reduce hydrolytic enzymes that cause polysorbate degradation. The addition of the EMPHAZE TM filter was evaluated in the common mAb purification process flow at two levels of processing. The first option was as a protein A load filter prior to protein A chromatography, where the HCCF was filtered prior to loading onto the protein A column. As summarized in Table 13, the relative hydrolytic activity was reduced by over 40% when the HCCF was filtered prior to protein A chromatography compared to the common purification process.

[0438] Table 13. Relative hydrolytic activity of compositions obtained from purification platforms.

[0439]

[0440] To demonstrate that the reduction in polysorbate degradation achieved using EMPHAZE TM is not related to a reduction in host cell proteins alone, the addition of EMPHAZE TMHCCF samples were passed through Protein A purification at fluxes and the CHOP and polysorbate degradation activity of the pool analyzed. As shown in Table 13, the reduction in CHOP values was dependent on the EMPHAZE Figure 9 TM filtering flux, while the significant reduction in polysorbate degradation rate was relatively constant. While CHOP values continued to increase with increasing EMPHAZE TM clarification flux, the significant reduction in polysorbate degradation specific activity achieved remained relatively stable compared to the control, up to 800 L / m 2 flux.

[0441] The second option evaluated was to place the EMPHAZE TM and X0SP depth filters downstream of the virus inactivation step, or as an upsample filter for the 2nd column chromatography. To perform this evaluation, Protein A pools from multiple molecules were neutralized to pH 5.5 or pH 8.0 and filtered through the EMPHAZE TM or X0SP to 300 L / m 2 flux. The polysorbate hydrolysis activity of the filtered pools was compared to control unfiltered samples. As summarized in Table 14, both the EMPHAZE TM and X0SP filters showed a significant reduction in polysorbate degradation compared to the unfiltered control pool. As shown in Table 14, the polysorbate hydrolysis activity (measured using a lipase activity assay in the anti-Tau mAb purification of the Protein A pool) was dependent on the filtering flux of the X0SP depth filter at pH 5.5. Figure 10

[0442] Table 14. Hydrolysis activity of compositions obtained from purification platforms.

[0443]

[0444]

[0445] The use of HIC membranes was evaluated as one approach to reduce polysorbate degradation. HIC membranes can be placed after the last polypeptide chromatography column step (e.g., anion exchange chromatography), after a pH hold step (e.g., pH 5-6) performed prior to virus filtration, and / or after a virus filtration step performed prior to a UFDF step.

[0446] The primary HIC membrane evaluated in this study was a phenyl membrane. The last polypeptide chromatography pool was filtered through the membrane filter after adjustment to different pH values. The relative polysorbate degradation of the filtered pool was analyzed. As shown in Table 15, the polysorbate hydrolysis activity (measured using a lipase activity assay in the anti-Tau mAb purification of the Protein A pool) was dependent on the filtering flux of the HIC membrane at pH 5.5. Figure 11A to Figure 11C ​​​The relative polysorbate activity is shown to be significantly reduced compared to control. The same data also show that the reduction in activity is dependent on membrane volume flux Figure 11A to Figure 11C Specifically, Figure 11A The specific activity of ocrelizumab polysorbate degradation at different fluxes at pH 5.5 is shown. Specifically, Figure 11B The specific activity of sarilumab polysorbate degradation at different fluxes at pH 5.5 is shown. Specifically, Figure 11B The specific activity of tocilizumab polysorbate degradation at different fluxes at pH 6.5 is shown Figure 11C .

[0447] Materials and Methods

[0448] Resins Pro A FF, TMAE and ceramic hydroxyapatite resins were purchased from GE Healthcare (Uppsala, Sweden), TOSOH Biosciences (King of Prussia, PA), and Bio-Rad (Hercules, California), respectively. Amicon centrifugal filters and X0SP depth filters were obtained from Millipore (Bedford, MA). EMPHAZE TM AEX depth filters were obtained from 3M (Meriden, CT), and Phenyl membranes were obtained from Sartorius (Bohemia, NY). 4-methylumbelliferyl caprylate, Triton TM X-100, and gum arabic were obtained from Research Organcis (Cleveland, OH) and Acros Organics (Bridgewater, NJ), respectively. Super refined (SR) grade PS20 for lipase activity assays was obtained from Croda (Newark, NJ). All monoclonal antibodies reported here are humanized or human IgGl expressed in CHO cells and manufactured by Roche (South San Francisco or Oceanside, CA).

[0449] For small EMPHAZE TM and other depth filters, a 25 cm 2 size capsule was used. The filters were first rinsed with 25 mM Tris, 250 mM NaCl pH 7.5 at a flow rate of 8 ml / min for 100 L / m2. After equilibration, the HCCF (Pro A load) or neutralized protein A elution pool was filtered to 800 L / m 2 per 100 L / m2 Fractions were collected and purified by subsequent column steps, and polysorbate degradation activity was measured.

[0450] for Phenyl membrane evaluation was performed using a 3 ml apparatus. The membrane was first washed with 30 ml of equilibration buffer at a flow rate of 15 L / min. After equilibration, the final chromatographic pooling was... Phenyl membrane filtration was performed, fractions were collected, and relative polysorbate degradation activity was determined.

[0451] All small-scale chromatography runs were performed on a 0.66 cm x 20 cm column. 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 the polysorbate degradation activity was determined.

[0452] Lipase activity was detected by cleavage of ester bonds in an umbrella ketone substrate with a polysorbate-like structure. In this study, lipase activity was determined by reacting 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). TM The reaction mixture was prepared by mixing X-100 and 0.1% gum arabic. Fluorescence excitation and emission wavelengths were set to 355 nm and 460 nm, respectively. Fluorescence kinetics were continuously monitored at 37 °C for 2–4 hours. The lipase activity of each sample was determined by calculating the initial reaction rate after kinetic curing using linear fitting, and 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.

[0453] 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 acetate-histidine solution, pH 5). The spiked samples were aseptically aliquoted into Eppendorf tubes and incubated at 25°C for up to 20 days. At each time point, aliquots were harvested and frozen at -70°C until free fatty acid extraction was performed.

[0454] Free fatty acids in each sample were extracted with an acetonitrile solution containing an isotopically labeled FFA internal standard. After centrifugation at 14,000 rpm for 5 minutes, the supernatant was transferred to an HPLC vial with a glass insert and frozen until measurement. Waters ACQUITY A Waters H-class BioUPLC system with a BEH300 C18 (1.7 μm, 2.1 x 150 mm) column was combined with an AB Sciex 6600 mass spectrometer for FFA detection. 5 mM ammonium acetate and 0.1% ammonium hydroxide aqueous solution were used as buffer A, and 100% acetonitrile as buffer B. The flow rate was 0.3 mL / min, and the column temperature was 60 °C. Free fatty acids were separated from a 5 μL sample. 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 within the linear range is fitted as a linear regression to calculate the initial hydrolysis rate.

[0455] Host cell proteins in samples were measured throughout the process using an internal CHO protein (CHOP) ELISA assay, and DNA was quantified using an internal qPCR method.

[0456] Example 6

[0457] This example demonstrates a comparison between two purification platforms used for antibody purification, which include: (1) deep filtration of HCCF using a 120ZB10A depth filter followed by protein A chromatography; or (2) using EMPHAZE. TM AEX deep filter was used, followed by protein A chromatography to further filter HCCF.

[0458] Harvest cell culture medium (HCCF) from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM3) was collected. 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). 2 Then, protein A chromatography was performed. Aliquots of the combined protein A chromatography 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 ratio of the aliquots after protein A chromatography to the FAMS rate is shown in [the table / image / image]. Figure 12An aliquot of the HCCF pool (prior to depth filtration) was used to obtain control measurements.

[0459] For the purification platform with EMPHAZE TM A purification platform (purification platform (2)) with an AEX depth filtration step, each HCCF pool was subjected to EMPHAZE TM AEX depth filtration (300 L / m 2 ), followed by protein A chromatography. An aliquot of the pool after protein A chromatography was collected. The aliquot was measured for polysorbate hydrolysis activity using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measured specific FAMS rate of the aliquot after protein A chromatography is shown in Figure 12 . An aliquot of the HCCF pool (prior to depth filtration) was used to obtain control measurements.

[0460] Example 7

[0461] This example demonstrates a comparison between the following two purification platforms for antibody purification, which include: (1) protein A chromatography of HCCF, followed by charcoal (40CR) filtration, followed by cation exchange chromatography using 50HS; or (2) protein A chromatography of HCCF, followed by depth filtration using an X0SP depth filter, followed by HIC using phenyl fast flow.

[0462] Harvest cell culture fluid (HCCF) was collected from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM4), respectively. For the purification platform without a depth filtration step (purification platform (1)), the three HCCF samples were subjected to protein A chromatography, respectively. The protein A chromatography pool was adjusted by adjusting the pH to 5.5 ± 0.3 with tris(hydroxymethyl)aminomethane (Tris) base, and then the adjusted pool was subjected to 40CR filtration (300 L / m 2 ), followed by 50 HS chromatography, respectively. An aliquot of the pool after 40CR filtration and 50HS filtration was collected. The aliquot was measured for polysorbate hydrolysis activity using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measured specific FAMS rate of the aliquot of the pool after 40CR filtration and 50HS chromatography is shown in Figure 13 and Figure 14 , respectively. An aliquot of the protein A chromatography pool (without 40CR filtration) was used to obtain control measurements.

[0463] For the purification platform with the X0SP depth filtration step (purification platform (2)), the three HCCF samples were each subjected to protein A chromatography. The protein A chromatography pool was adjusted by adjusting the pH to 5.5 ± 0.3 with tris(hydroxymethyl)aminomethane (Tris) base, and then the adjusted pool was each subjected to X0SP filtration (300 L / m 2 ), followed by phenyl flow chromatography. Aliquots of the X0SP filtered pool and the phenyl flow chromatography pool were collected. The aliquots were measured for polysorbate hydrolysis activity using FAMS, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measured FAMS rates for the aliquots of the X0SP filtered pool and the phenyl flow chromatography pool are shown in Figure 13 and Figure 14 , respectively. Aliquots of the protein A chromatography pool (without X0SP depth filtration) were used to obtain control measurements.

[0464] Example 8

[0465] This example demonstrates a comparison between the following three purification platforms for antibody purification, which include: (1) protein A chromatography of HCCF, followed by depth filtration using a PDD1 depth filter, followed by Capto Adhere multimodal chromatography; (2) protein A chromatography of HCCF, followed by depth filtration using an EMPHAZE TM depth filter, followed by Capto Adhere multimodal chromatography; or (3) protein A chromatography of HCCF, followed by depth filtration using a PDD1 depth filter, followed by Capto Adhere multimodal chromatography.

[0466] Harvest cell culture fluid (HCCF) was collected from three different cell cultures expressing three different antibody moieties (AM1, AM2, and AM4), respectively. For the purification platform without the depth filtration step (purification platform (1)), the three HCCF samples were each subjected to protein A chromatography. The protein A chromatography pool was adjusted by adjusting the pH to 8.0 ± 0.5 with Tris base, and then the adjusted pool was each subjected to 40CR filtration (300 L / m 2 ), followed by Capto Adhere chromatography. Aliquots of the 40CR filtered pool were collected. The aliquots were measured for polysorbate hydrolysis activity using LEAP assay, the method of which is disclosed in more detail in the Materials and Methods section of Example 1. The measured LEAP rates for the aliquots of the 40CR filtered pool are shown in Figure 15In the middle section, aliquots of protein A chromatographic pools (without 40 CR filtration) were used to obtain control measurements.

[0467] For those with EMPHAZE TM The purification platform for the deep filtration step (purification platform (2)) was used to perform protein A chromatography on the three HCCF samples. 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. TM Deep filtration (300L / m 2 Then, Capto Adhere chromatography was performed. EMPHAZE was collected. TM Aliquots of the merged product after deep filtration were prepared. The hydrolytic activity of polysorbate in the aliquots was measured using LEAP, a method disclosed in more detail in the Materials and Methods section of Example 1. EMPHAZE TM The measurement ratio of the aliquots of the merged material after depth filtration is shown in the LEAP rate. Figure 15 In the middle. Aliquots of protein A chromatographic pools were used (without EMPHAZE). TM (Deep filtering) is used to obtain control measurements.

[0468] 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 pools were adjusted to pH 8.0 ± 0.5 with Tris base, and then the adjusted pools were subjected to PDD1 deep filtration (300 L / m³). 2 Then, Capto Adhere chromatography was performed. Aliquots of the PDD1 deep-filtered merged product 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 ratio of the aliquots of the PDD1 deep-filtered merged product to the LEAP rate is shown in... Figure 15 In the middle section, aliquots of protein A chromatographic pools (without PDD1 depth filtration) were used to obtain control measurements.

[0469] Example 9

[0470] 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), where the workflows use different pre-C1 depth filters, followed by Merck Millipore... The pre-C2 filtration step of the HC Pro X0SP filter. The pre-C1 depth filter being compared is a depth filter containing synthetic materials with a defined chemical composition (3M).TM EMPHAZE TM AEX hybrid purifier) and harvest clarification depth filter (ZETA PLUS TM EXT ZB series, 120ZB). The 120ZB and EMPHAZE TM depth filters are positively charged, while the X0SP depth filter is negatively charged at pH > 4.5.

[0471] The experimental workflow and assignment of sample names are shown in Figure 16 Briefly, cell-free harvest fluid having a turbidity of about 80 NTU and containing TYRP1 TCB antibody was collected from a bioreactor and used as the loading material for the experiments. 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 TM depth filter. The two filtrates were then sterile filtered and subjected to protein A chromatography using MabSelect SuRe TM media from GE Healthcare. The eluates from the 120ZB depth filter workflow and the EMPHAZE TM depth filter workflow were separately titrated to pH 5.5. For each workflow, the titrated eluate was divided into aliquots; one sample was sterile filtered using a 0.2 pm sterile filter and retained as a reference, and a second aliquot (about 100 mL to about 120 mL) was filtered using a small Merck Millipore HC Pro X0SP filter (5 cm 2 filter area). From the 120ZB workflow, three eluate fractions from the X0SP filtration were collected. From the EMPHAZE TM workflow, four eluate fractions from the X0SP filtration were collected. Each X0SP eluate fraction was subsequently filtered using a 0.2 pm sterile filter.

[0472] Each resulting aliquot was then analyzed by LEAP assay according to the method provided in Example 1. Comparison of the LEAP assay results indicates that both pre-C1 depth filter workflows reduced hydrolytic activity well, and the X0SP filter significantly reduced the hydrolytic activity of the capture column output Figure 17 ). Comparison of the 120ZB and EMPHAZE TM workflows indicates that the A-protein eluate from the EMPHAZE TM filtered material has 33% less hydrolytic activity than the A-protein eluate from the 120ZB filtered material Figure 17 ).

[0473] Example 10

[0474] This example demonstrates an evaluation and comparison of purifications of different HCCF samples containing TYRP1 TCB antibodies using a purification platform comprising an X0SP depth filtration step on the Protein A chromatography eluate (such as disclosed in PCT / EP2019 / 08614, which is incorporated herein in its entirety by reference).

[0475] 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 TM Media. The eluates (CF 238 MSS eluate and CF 239 MSS eluate) were each titrated to pH 5.5 with 1 M TRIS / HCl (pH 9.0) and then subjected to X0SP depth filtration. Specifically, 35 L of the CF 238 MSS eluate or 20 L of the CF 239 MSS eluate was filtered through an X0SP filter (1 m 2 or 0.55 m 2 ) at a flow rate of 160 L / m 2 / h, respectively. Meanwhile, aliquots of the CF 238 and CF 239 HCCF samples were each purified using a reference purification platform that did not include an X0SP depth filtration step after Protein A chromatography.

[0476] Lipase activity was performed on each of the resulting eluates as discussed in Example 1. The results of the lipase activity obtained from the reference and the purification platform including the X0SP depth filter for both CF 238 and CF 239 are shown in Figure 18A (CF 238) and Figure 18B (CF 239). As shown in Figure 18A and Figure 18B , the hydrolytic activity of the eluates from the purification platform including X0SP was significantly reduced.

[0477] Example 11

[0478] This example demonstrates a comparison of purifications of plasminogen using four purification platforms that incorporate different depth filtration steps for filtering the eluate from an adjusted affinity chromatography (Protein A chromatography). 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.

[0479] Adjust the affinity chromatography eluate to pH 6.0 ± 0.2 using 2M Tris. Equilibrate the PDD1 and XSOP filters with at least 220 mL of the appropriate buffer (25 mM Tris / acetate). Load <200 L / mL onto both filters. 2 The flow rate for both the PDD1 and XS0P filters was 11 ml / min. Pressure was controlled during the experiment. The eluent from the depth filter was 100 L / min. 2 and 200L / m 2 Then it is classified.

[0480] 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 of 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 the HCP measurement are shown in [the table / information]. Figure 19B middle.

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, in the following order: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A depth filtering step, which includes processing via a depth filter. The depth filter mentioned above is an EMPHAZE™ depth filter. This reduces the enzyme hydrolysis activity rate of the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step, wherein the enzyme hydrolysis activity rate is the polysorbate hydrolysis activity rate.

2. The method of claim 1, wherein the relative reduction in the rate of enzyme hydrolysis activity of the composition is at least 20% compared to purifying the sample using the same purification platform without the depth filtration step.

3. 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 the following order: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A depth filtering step, which includes processing via a depth filter. The depth filter mentioned above is an EMPHAZE™ depth filter. This reduces the level of the hydrolytic enzymes in the composition compared to purifying the sample using the same purification platform without the depth filtration step, wherein one or more of the hydrolytic enzymes are capable of hydrolyzing polysorbate.

4. The method of claim 3, wherein the relative reduction in the level of one or more hydrolytic enzymes in the composition is at least 20% compared to purifying the sample using the same purification platform without the depth filtration step.

5. 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 the following order: (a) A capture step, the capture step comprising processing by affinity chromatography; as well as (b) A depth filtering step, which includes processing via a depth filter. The depth filter mentioned above is an EMPHAZE™ depth filter. 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.

6. The method of claim 5, wherein the relative reduction in degradation of the polysorbate in the composition is at least 5% compared to purifying the sample using the same purification platform without the depth filtration step.

7. The method according to any one of claims 1-6, wherein the purification platform is used to purify the target from the sample, wherein the sample comprises the target and one or more host cell impurities.

8. The method of claim 7, wherein the target comprises a polypeptide.

9. The method according to claim 7 or 8, wherein the host cell impurity is a host cell protein.

10. The method according to any one of claims 1-9, wherein the affinity chromatography is selected from the group consisting of protein A chromatography, protein G chromatography, protein L chromatography, XL chromatography and κ chromatography.

11. The method according to any one of claims 1-9, wherein the affinity chromatography is selected from the group consisting of FcXL chromatography and κXL chromatography.

12. The method according to any one of claims 1-11, wherein the purification platform further comprises a virus inactivation step, wherein the virus inactivation step is performed after the capture step.

13. The method of claim 12, wherein the deep filtering step is performed after the virus inactivation step.

14. The method according to any one of claims 1-13, wherein the purification platform further comprises a second depth filtration step performed prior to the capture step.

15. The method of claim 14, wherein the second depth filtering step comprises processing through a second depth filter.

16. The method of claim 15, wherein the second 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.

17. The method of claim 16, wherein at least a portion of the substrate of the second depth filter comprises surface modification.

18. The method according to claim 17, wherein the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification and anionic surface modification.

19. The method according to any one of claims 15-18, wherein the second depth filter is selected from the group consisting of an EMPHAZE™ depth filter, a PDD1 depth filter, a ZETA PLUS™ 120ZA depth filter, a ZETA PLUS™ 120ZB depth filter, and a VR02 depth filter.

20. The method according to any one of claims 1-19, wherein the purification platform further comprises a sterilization step performed before the capture step and after, if present, a second depth filtration step, wherein the sterilization step comprises treatment with a sterile filter.

21. The method according to any one of claims 1-20, 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.

22. The method of claim 21, wherein the one or more purification steps include a peptide purification step.

23. The method of claim 21 or 22, wherein the purification platform further comprises another depth filtration step performed before, between or after the one or more purification steps.

24. The method according to any one of claims 1-23, wherein the purification platform further comprises an ultrafiltration / percolation (UFDF) step, and wherein the UFDF step is performed after the one or more purification steps.

25. The method of claim 24, wherein the purification platform further comprises another depth filtration step performed before or after the UFDF step.

26. The method according to any one of claims 1-25, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step.

27. The method of claim 26, wherein the HIC purification step is performed before, between, or after, if any, the one or more purification steps.

28. The method of claim 26, wherein the HIC purification step is performed after the one or more purification steps and before the UFDF step, if present.

29. The method of claim 24 or 25, wherein the purification platform further comprises a pH maintenance step, wherein the pH maintenance step is performed after, if present, the one or more purification steps and before the UFDF step.

30. The method of claim 29, wherein the purification platform further comprises a virus filtration step, wherein the virus filtration step is performed after the pH maintenance step and before the UFDF step.

31. The method of claim 30, wherein the virus filtering step comprises processing through a virus filter.

32. The method of claim 26, wherein the HIC purification step comprises processing through a HIC filter.

33. The method according to any one of claims 21-32, wherein the one or more purification steps each independently comprise treatment by chromatography selected from the group consisting of: anion exchange chromatography, cation exchange chromatography, hydrophobic charge-induced chromatography, ceramic hydroxyapatite chromatography, and multi-component chromatography.

34. The method according to any one of claims 21-33, 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.

35. 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) A depth filtering step, the depth filtering step including processing through a depth filter; (d) Second polypeptide purification step; (e) The third polypeptide purification step; and (f) Ultrafiltration / percolation (UFDF) steps, The depth filter mentioned above is an EMPHAZE™ depth filter. This reduces the enzyme hydrolysis activity rate of the composition compared to purifying the sample using the same purification platform without the aforementioned depth filtration step, wherein the enzyme hydrolysis activity rate is the polysorbate hydrolysis activity rate.

36. The method of claim 35, wherein the purification platform further comprises, 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.

37. The method of claim 36, wherein the virus filtering step comprises processing through a virus filter.

38. The method according to any one of claims 35-37, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step performed during one or more of the following: (i) after the third polypeptide purification step and before the pH maintenance step; (ii) after the pH maintenance step and before the virus filtration step; or (iii) After the virus filtering step and before the UFDF step.

39. 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) First depth filtering step; (b) A capture step, the capture step comprising processing by affinity chromatography; (c) Virus inactivation procedures; and (d) A second depth filtering step, the second depth filtering step including processing through a depth filter; The depth filter in the second depth filtering step is an EMPHAZE™ depth filter. This reduces the enzyme hydrolysis activity rate of the composition compared to purifying the sample using the same purification platform without the second depth filtration step, wherein the enzyme hydrolysis activity rate is the polysorbate hydrolysis activity rate.

40. The method of claim 39, wherein the first depth filtering step comprises processing via a depth filter.

41. The method of claim 40, wherein the depth filter of the first depth filtration step 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.

42. The method of claim 41, wherein at least a portion of the substrate of the depth filter in the first depth filtering step comprises surface modification.

43. The method according to claim 42, wherein the surface modification is one or more of quaternary ammonium surface modification, cationic surface modification and anionic surface modification.

44. The method according to any one of claims 41-43, wherein the depth filter in the first depth filtering step is selected from the group consisting of an EMPHAZE™ depth filter, a PDD1 depth filter, a ZETA PLUS™ 120ZA depth filter, a ZETA PLUS™ 120ZB depth filter, and a VR02 depth filter.

45. The method according to any one of claims 39-44, wherein the purification platform further includes a sterilization step performed after the first depth filtration step and before the capture step, wherein the sterilization step includes treatment with a sterile filter.

46. ​​The method according to any one of claims 1-45, further comprising determining the rate of enzyme hydrolysis activity of the composition.

47. The method according to any one of claims 1-46, further comprising determining the level of one or more hydrolytic enzymes in the composition.

48. The method according to any one of claims 1-47, wherein the composition comprises polysorbate.

49. The method of claim 48, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

50. The method according to any one of claims 1-49, further comprising a sample processing step.

51. The method according to any one of claims 1-50, wherein the sample is or is derived from a cell culture sample.

52. The method of claim 51, wherein the cell culture sample comprises host cells, and wherein the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli cells.

53. The method according to any one of claims 1-52, wherein the sample comprises a host cell or a component derived from the host cell.

54. The method according to any one of claims 1-53, wherein the sample comprises one or more host cell proteins, and wherein one or more host cell proteins is a hydrolase.

55. The method according to claim 54, wherein the hydrolytic enzyme is a lipase, a thioesterase, a phospholipase, or a ceramide enzyme.

56. The method of claim 54, wherein the hydrolase is an esterase.

57. The method according to any one of claims 1-56, wherein the sample comprises a target, and wherein the target is an antibody moiety.

58. The method of claim 57, wherein the antibody portion is a monoclonal antibody.

59. The method of claim 57 or 58, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

60. The method according to any one of claims 57-59, 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.

61. The method according to any one of claims 57-60, 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.

62. A method for generating a pharmaceutical composition, comprising obtaining the composition from the purification platform of any one of claims 1-61.

63. The method of claim 62, wherein the composition comprises an antibody moiety and polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, and wherein the composition has a shelf life of more than 24 months.

64. The method of claim 62, wherein the composition comprises an antibody moiety 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 moiety composition, wherein the shelf life is extended by at least 6 months compared to the shelf life specified in the document.

65. The method of claim 62, wherein the composition comprises an antibody portion, wherein the composition has reduced polysorbate degradation, wherein the degradation is reduced by at least 20% compared to the degradation specified in documents submitted to health authorities relating to the composition.

66. The method of claim 62, wherein the composition comprises an antibody portion and a polysorbate, wherein the polysorbate degrades by 20% or less per year during storage of the liquid composition.

67. The method according to any one of claims 63-66, wherein the antibody portion is a monoclonal antibody.

68. The method according to any one of claims 63-67, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

69. The method according to any one of claims 63-68, 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.

70. The method according to any one of claims 63-69, 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.

71. The method according to any one of claims 63-70, wherein the hydrolysis activity rate of the polysorbate is reduced by at least 20%.

72. The method according to any one of claims 63-71, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

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