A method for reducing the enzymatic hydrolysis rate in a composition obtained from a purification platform.

Abstract

This invention provides an improved method for purifying biotherapeutic products produced from host cell cultures for pharmaceutical use. [Solution] A purification platform comprising a deep filtration step and / or a hydrophobic interaction chromatography (HIC) step is provided. Methods of using the purification platform and compositions obtained therefrom, such as pharmaceutical compositions, are also provided. In some embodiments, methods are provided for reducing the enzymatic hydrolysis rate of compositions obtained from the purification platform, where the enzymatic hydrolysis rate is the enzymatic polysorbate hydrolysis rate.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Application No. 62 / 961,609 filed on 15 January 2020 and U.S. Provisional Application No. 62 / 843,261 filed on 3 May 2019, which are incorporated herein by reference in their entirety.

[0002] This disclosure provides a purification platform comprising a deep filtration step and / or a hydrophobic interaction chromatography (HIC) step. Methods using the purification platform described herein and compositions obtained therefrom are also disclosed herein. [Background technology]

[0003] Biotherapeutic products, such as antibodies, produced from host cell cultures require purification to remove host cell proteins and other impurities that may affect product quality and therapeutic efficacy. Current purification methods do not remove all host cell proteins, including host cell hydrolases, and impurities. Therefore, impurities remaining with host cell proteins and the purified target may affect the purified target itself as well as other additives, such as surfactants and other components added for formulation purposes. Consequently, improved methods are needed for purifying biotherapeutic products produced from host cell cultures for pharmaceutical use.

[0004] All references cited herein, including patent applications and publications, are incorporated in their entirety by reference. [Overview of the Initiative]

[0005] In one embodiment, a method is provided for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, comprising subjecting a sample to a purification platform including (a) a capture step and (b) a deep filtration step, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0006] In some embodiments, the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate. In some embodiments, the relative decrease in the enzyme hydrolysis activity rate of the composition compared to the purification of a sample using the same purification platform without a deep filtration step is at least about 20%.

[0007] In another embodiment, a method is provided for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, comprising subjecting a sample to the purification platform which includes (a) a capture step and (b) a deep filtration step, thereby reducing the level of hydrolytic enzymes in the composition compared to the purification of a sample using the same purification platform which does not include the deep filtration step. In some embodiments, the one or more hydrolytic enzymes are capable of hydrolyzing polysorbate. In some embodiments, the relative reduction in the level of one or more hydrolytic enzymes in the composition compared to the purification of a sample using the same purification platform which does not include the deep filtration step is at least about 20%.

[0008] In another embodiment, a method is provided for reducing the degradation of polysorbate in a composition obtained from a purification platform, comprising subjecting a sample to the purification platform which includes (a) a capture step and (b) a deep filtration step, thereby reducing the degradation of polysorbate in the composition compared to the purification of the sample using the same purification platform which does not include the deep filtration step. In some embodiments, the relative reduction in the degradation of polysorbate in the composition compared to the purification of the sample using the same purification platform which does not include the deep filtration step is at least about 5%.

[0009] In some embodiments, the purification platform is for purifying a target from a sample, and the sample contains the target and one or more host cell impurities. In some embodiments, the target contains a polypeptide. In some embodiments, the host cell impurities are host cell proteins.

[0010] In some embodiments, the deep filtration step is performed before the capture step, or the deep filtration step is performed after the capture step.

[0011] In some embodiments, the deep filtration process includes treatment with a deep filter. In some embodiments, the deep filter includes a substrate comprising one or more of the following: a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a cohesive resin, and an ash composition. In some embodiments, at least a portion of the substrate of the deep filter includes surface modification. In some embodiments, the surface modification is one or more of quaternary amine surface modification, cationic surface modification, and anionic surface modification. In some embodiments, the deep filter is selected from the group consisting of EMPHAZE® deep filter, X0SP deep filter, PDD1 deep filter, ZETA PLUS® 120ZA deep filter, and ZETA PLUS® 120ZB deep filter.

[0012] In some embodiments, the capture step includes processing by affinity chromatography. 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, FcXL chromatography, protein XL chromatography, kappa chromatography, and kappa XL chromatography.

[0013] In some embodiments, the purification platform further includes a virus inactivation step, which is performed after the capture step. In some embodiments, the depth filtration step is performed after the virus inactivation step.

[0014] In some embodiments, the purification platform further includes another depth filtration step that is performed before the capture step.

[0015] In some embodiments, the purification platform further includes one or more purification steps, which are performed after the capture step, the depth filtration step, and the virus inactivation step if present. In some embodiments, the one or more purification steps include a polypeptide purification step. In some embodiments, the purification platform further includes another depth filtration step that is performed before, during, or after the one or more purification steps.

[0016] In some embodiments, the purification platform further includes an ultrafiltration / diafiltration (UFDF) step, which is performed after the one or more purification steps. In some embodiments, the purification platform further includes another depth filtration step that is performed before or after the UFDF step.

[0017] 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, during, 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.

[0018] In some embodiments, the purification platform further includes a pH holding step, which 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 viral filtration step, which is performed after the pH holding step and before the UFDF step. In some embodiments, the viral filtration step includes treatment with a viral filter.

[0020] In some embodiments, the HIC purification step includes treatment with an HIC filter.

[0021] In some embodiments, one or more purification steps independently include treatment 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, one or more purification steps independently include treatment by chromatography selected from the group consisting of: DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel®, Capto MMC, and Capto Adhere.

[0022] In another embodiment, a method is provided for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order: (a) a capture step including treatment by affinity chromatography; (b) a virus 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 deep filtration step carried out at one or more of the following locations: (i) before the capture step; (ii) after the capture step and before the virus inactivation step; (iii) after the virus inactivation step and before the second polypeptide purification step; (iv) after the second polypeptide purification step and before the third polypeptide purification step; or (v) after the third polypeptide purification step and before the ultrafiltration / diafiltration (UFDF) step, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0023] In some embodiments, the purification platform further includes a pH holding step and a virus filtration step, which are performed after the third polypeptide purification step and before the UFDF step, in the following order. In some embodiments, the virus filtration step includes treatment with a virus filter.

[0024] In some embodiments, the purification platform further includes a hydrophobic interaction chromatography (HIC) purification step carried out in one or more of the following: (i) after a third polypeptide purification step and before a pH holding step, (ii) after a pH holding step and before a viral filtration step, or (iii) after a viral filtration step and before a UFDF step.

[0025] In some embodiments, the method further includes determining the enzymatic hydrolysis rate of the composition.

[0026] In some embodiments, the method further includes determining the level of one or more hydrolytic enzymes in the composition.

[0027] In some embodiments, the composition includes a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0028] In some embodiments, the method further includes a sample preparation step.

[0029] In some embodiments, the sample is a cell culture sample or derived from a cell culture sample. In some embodiments, the cell culture sample includes host cells, which are Chinese hamster ovary (CHO) cells or Escherichia coli cells. In some embodiments, the sample includes host cells or components derived therefrom. In some embodiments, the sample includes one or more host cell proteins, one of which is a hydrolase.

[0030] In some embodiments, the hydrolytic enzyme is a lipase, esterase, thioesterase, phospholipase, or ceramidase.

[0031] In some embodiments, the sample includes a target, and the target is an antibody portion. In some embodiments, the antibody portion is a monoclonal antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0032] In some embodiments, the antibody moiety 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-α-beta 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.

[0033] In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0034] In another embodiment, a pharmaceutical composition obtained from any one of the methods described herein is provided.

[0035] In another embodiment, a formulated antibody partial composition comprising polysorbate is provided, wherein the polysorbate hydrolysis activity rate is reduced and the composition has a shelf life of more than 24 months.

[0036] In another embodiment, a formulated antibody partial composition is provided, comprising an antibody portion and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, and the shelf life of the composition is extended compared to the shelf life indicated in a document submitted to the health authorities relating to the formulated antibody partial composition, and the shelf life is extended by at least 6 months compared to the shelf life indicated in the said document.

[0037] In another embodiment, a formulated antibody partial composition is provided, comprising an antibody moiety, wherein the formulated antibody partial composition exhibits reduced polysorbate degradation, the degradation being at least about 20% lower than the degradation described in the documentation submitted to the health authorities relating to the formulated antibody partial composition.

[0038] In another embodiment, a formulated antibody partial composition is provided, comprising an antibody portion and a polysorbate, wherein the polysorbate degrades by 20% or less per year during storage of the liquid composition.

[0039] In some embodiments, the antibody portion of the formulated antibody partial composition is a monoclonal antibody. In some embodiments, the antibody portion of the formulated antibody partial composition is a human antibody, a humanized antibody, or a chimeric antibody.

[0040] In some embodiments, the antibody portion of the formulated antibody partial composition is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α-beta 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.

[0041] In some embodiments, the antibody portion of the formulated antibody portion composition is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0042] In some embodiments, the polysorbate hydrolysis activity rate of the formulated antibody partial 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.

[0043] In another embodiment, a method is provided for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order, (a) a capture step comprising treatment by affinity chromatography, and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step, or after the capture step and before the purification step, each deep filtration step comprising treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membranes, and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps. In some embodiments, the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate. In some embodiments, the relative decrease in the enzyme hydrolysis activity rate of the composition compared to the purification of a sample using the same purification platform without a deep filtration step is at least about 20%.

[0044] In another embodiment, a method is provided for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order, a capture step comprising treatment by affinity chromatography, and a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step and before the purification step, or after the purification step, and each deep filtration step comprises treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone 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 the purification of a sample using the same purification platform without one or more deep filtration steps. In some embodiments, one or more hydrolytic enzymes can hydrolyze polysorbate. In some embodiments, the relative reduction in the level of one or more hydrolytic enzymes in the composition compared to the purification of a sample using the same purification platform without a deep filtration step is at least about 20%.

[0045] In another embodiment, a method is provided for reducing the degradation of polysorbate in a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order, a capture step comprising treatment by affinity chromatography, and a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step, or after the capture step and before the purification step, each deep filtration step comprising treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membranes, and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the degradation of polysorbate in the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps. In some embodiments, the relative reduction in polysorbate degradation in the composition compared to the purification of a sample using the same purification platform without a deep filtration step is at least about 5%.

[0046] In some embodiments, the deep filter containing silica and polyacrylic fibers includes a silica filter aid and polyacrylic fiber pulp.

[0047] In some embodiments, a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane comprises four layers comprising the hydrogel Q-functionalized nonwoven fabric material and a 9-zone microporous membrane.

[0048] In some embodiments, a deep filter comprising cellulose fibers, diatomaceous earth, and perlite comprises two layers, each layer comprising a cellulose filter matrix, the cellulose filter matrix being impregnated with a filtration aid comprising one or more diatomaceous earth or perlite, and each layer further comprising a resin binder.

[0049] In some embodiments, the deep filter is selected based on the pH of the solution entering the deep filter. In some embodiments, a deep filter comprising silica and polyacrylic fibers is selected when the solution entering the deep filter has a pH of about 5 to about 6.5. In some embodiments, a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane is selected when the solution entering the deep filter has a pH of about 7 to about 8.5. In some embodiments, the method further includes the step of selecting the deep filter based on the pH of the solution entering the deep filter.

[0050] In some embodiments, the purification platform sequentially includes a deep filtration step, which includes treatment with a hydrogel Q-functionalized nonwoven fabric medium and a deep filter containing a multizone microporous membrane; a capture step, which includes treatment with protein A chromatography; and a purification step.

[0051] In some embodiments, the purification step includes treatment with HIC. In some embodiments, HIC is phenylSEPHAROSE® high-speed flow chromatography.

[0052] In some embodiments, the purification step includes treatment by cation exchange chromatography. In some embodiments, the cation exchange chromatography is POROS® 50HS.

[0053] In some embodiments, the purification platform further includes a second deep filtration step, which involves treatment with a deep filter containing silica and polyacrylic fibers, the second deep filtration step being performed after the capture step and before the purification step.

[0054] In some embodiments, the purification step includes treatment by multimodal chromatography. In some embodiments, the multimodal chromatography is CaptoAdhere.

[0055] In some embodiments, the purification platform further includes a second deep filtration step, which involves processing with a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane, the second deep filtration step being performed after the capture step and before the purification step.

[0056] In some embodiments, the purification platform is for purifying a target from a sample, and the sample contains the target and one or more host cell impurities. In some embodiments, the target contains a polypeptide. In some embodiments, the host cell impurities are host cell proteins.

[0057] In some embodiments, the purification platform further includes a virus inactivation step, which is performed after the capture step. In some embodiments, one or more deep filtration steps are performed after the virus inactivation step.

[0058] In some embodiments, the purification platform further includes an ultrafiltration / diafiltration (UFDF) step, which is performed after the purification step.

[0059] In some embodiments, the methods described herein further include determining the enzymatic hydrolysis activity rate of the composition.

[0060] In some embodiments, the methods described herein further include determining the level of one or more hydrolytic enzymes in the composition.

[0061] In some embodiments, the composition includes a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0062] In some embodiments, the method described herein further includes a sample preparation step.

[0063] In some embodiments, the sample is a cell culture sample or derived from a cell culture sample. In some embodiments, the cell culture sample includes host cells, which are Chinese hamster ovary (CHO) cells or E. coli cells. In some embodiments, the sample includes host cells or components derived therefrom.

[0064] In some embodiments, the sample comprises one or more host cell proteins, one of which is a hydrolase. In some embodiments, the hydrolase is a lipase, esterase, thioesterase, phospholipase, or ceramidase. In some embodiments, the sample comprises a target, which is an antibody moiety. In some embodiments, the antibody moiety is a monoclonal antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody moiety 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-α-beta 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 moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0065] In another embodiment, pharmaceutical compositions obtained from any of the methods described herein are provided.

[0066] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present disclosure is further illustrated by the following embodiments, but these embodiments should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described therein. [Brief explanation of the drawing]

[0067] [Figure 1A] This figure shows an exemplary process of the purification platform 100. [Figure 1B] This figure shows exemplary options for the purification platform process.

[0068] [Figure 2] This figure shows a bar graph of the amount of free fatty acids measured in compositions obtained from a purification platform using the FAMS assay.

[0069] [Figure 3] This figure shows a bar graph of hydrolysis activity measured using a lipase activity assay with compositions obtained from a purification platform.

[0070] [Figure 4] This figure shows a bar graph of the amount of free fatty acids measured in compositions obtained from a purification platform using the FAMS assay.

[0071] [Figure 5] A schematic diagram of the purification options for falisimab is shown.

[0072] [Figure 6A] This figure shows a bar graph of PS20 hydrolysis activity measured in the FcXL eluate for falisimab before filtration with a PDD1 filter (Figure 6A). [Figure 6B] This figure shows a bar graph of PS20 hydrolysis activity measured in the FcXL eluate for falisimab after filtration with a PDD1 filter (Figure 6B).

[0073] [Figure 7] This figure shows a bar graph of the amount of free fatty acids measured in compositions obtained from a purification platform using the FAMS assay.

[0074] [Figure 8A] This figure shows a bar graph of hydrolysis activity measured in the protein A eluate after filtration using a deep filter. [Figure 8B] This figure shows a bar graph of hydrolysis activity measured in the protein A eluate after filtration using a deep filter.

[0075] [Figure 9] This figure shows the relative levels of CHOP and polysorbate degradation activity in the protein A eluate after clarification with EMPHAZE® deep filter.

[0076] [Figure 10] This figure shows the specific activity of polysorbate degradation of compositions obtained from an X0SP deep filter (pH 5.5) at different processing rates.

[0077] [Figure 11A] This figure shows the specific activity of ocrelizumab at pH 5.5 for polysorbate degradation at different treatment doses. [Figure 11B] This figure shows the specific activity of sericrelumab at pH 5.5 for polysorbate degradation at different treatment doses. [Figure 11C] This figure shows the specific activity of tocilizumab at pH 6.5 for polysorbate degradation at different treatment doses.

[0078] [Figure 12] This figure shows a bar graph of the specific FAMS ratio of compositions obtained from the purification platform.

[0079] [Figure 13]This figure shows a bar graph of the specific FAMS ratio of compositions obtained from the purification platform.

[0080] [Figure 14] This figure shows a bar graph of the specific FAMS ratio of compositions obtained from the purification platform.

[0081] [Figure 15] This figure shows a bar graph of the specific LEAP rate of compositions obtained from the purification platform.

[0082] [Figure 16] This figure shows a schematic diagram of the purification workflow.

[0083] [Figure 17] This figure shows a bar graph of the average conversion rate of compositions obtained from the purification platform, measured using the LEAP assay.

[0084] [Figure 18A] This figure shows a bar graph of the hydrolysis activity of compositions obtained from the purification platform, measured using a lipase activity assay. [Figure 18B] This figure shows bar graphs of the hydrolytic activity of compositions obtained from the purification platform, measured using a lipase activity assay. Figure 18A shows the results obtained from CF238. Figure 18B shows the results obtained from CF239.

[0085] [Figure 19A] This figure shows a bar graph of the average conversion rate of compositions obtained from the purification platform, measured using the LEAP assay. [Figure 19B] This figure shows a bar graph of the hydrolysis activity of compositions obtained from the purification platform, measured using a lipase activity assay. [Modes for carrying out the invention]

[0086] In some embodiments, this application provides a method for purifying a target from a sample containing the target, comprising the step of subjecting the sample to a purification platform disclosed herein, which includes one or more deep filtering steps and / or one or more hydrophobic interaction chromatography (HIC) steps.

[0087] This disclosure is based in part on the unexpected finding that a purification platform including one or more deep filtration steps, such as a deep filtration step performed on host cell culture medium (HCCF) and / or affinity chromatography eluate, reduces the enzymatic hydrolysis rate of the composition obtained therefrom. Furthermore, this disclosure is based in part on the unexpected finding that a purification platform including one or more HIC steps reduces the enzymatic hydrolysis rate of the composition obtained therefrom, and that a purification platform including both a deep filtration step and a HIC step may further reduce the enzymatic hydrolysis rate of the composition obtained therefrom.

[0088] Those skilled in the art will understand that modifications to the forms and details of the embodiments described herein can be made without departing from the scope of this disclosure. Furthermore, although various advantages, aspects, and objectives have been described with reference to various embodiments, the scope of this disclosure should not be limited by reference to such advantages, aspects, and objectives. definition

[0089] For the purposes of interpreting this Spec., the following definitions shall apply, and wherever appropriate, a term used in the singular form shall also include the plural form, and conversely, a term used in the plural form shall also include the singular form. In the event of any conflict between any of the following definitions and any document incorporated herein by reference, the following definition shall prevail.

[0090] The term “antibody portion” includes the full-length antibody and its antigen-binding fragment. In some embodiments, the 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 of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC)CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC)CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by convention of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are more conserved than the CDRs themselves and interpose between adjacent stretches known as framework regions (FRs) that form a scaffold for supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a semi-synthetic antibody. In some embodiments, the antibody portion is a diabody. In some embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. In some embodiments, the antibody moiety is linked to a fusion protein. In some embodiments, the antibody moiety is bound to an immunostimulatory protein such as an interleukin. In some embodiments, the antibody moiety is linked to a protein that facilitates entry across the blood-brain barrier.

[0091] As used herein, the term “antigen-binding fragment” refers to an antibody fragment that includes, for example, a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen to which a parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, an antigen-binding fragment may include one or more CDRs derived from a particular human antibody grafted onto a framework region derived from one or more different human antibodies.

[0092] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody belonging to a particular species or antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody belonging to a different species or antibody class or subclass, and includes fragments of such antibodies insofar as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0093] As used herein, the term "multispecific antibody" refers to a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has two binding specificities (a bispecific antibody). In certain embodiments, a multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0094] With respect to an antibody or antibody moiety, the term "semi-synthetic" means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more unnaturally occurring (i.e., synthetic) sequences.

[0095] "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly bound noncovalently. The folding of these two domains creates six hypervariable loops (three from the heavy chain and three from the light chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0096] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure desirable for antigen binding. For an overview of scFv, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0097] The term "diabody" refers to a small antibody fragment prepared by constructing an scFv fragment (see previous paragraph) between the VH and VL domains with a short linker (approximately 5-10 residues) such that interchain pairing, rather than intrachain pairing, is achieved in the V domain, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two "cross-resolved" scFv fragments, where the VH and VL domains of two antibodies reside on different polypeptide chains. Diabodies are described in more detail, for example, European Patent No. 404,097, International Publication No. 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0098] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains a minimal amount of sequences derived from the non-human antibody. Mostly, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired antibody specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications further improve the antibody's performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also, in some cases, include the constant region (Fc) of immunoglobulins, typically at least a portion of the constant region of human immunoglobulins. For further details, see, for example, 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).

[0099] In some embodiments, the methods described herein include one or more deep filtration steps. The deep filtration step is a chromatographic technique that includes treatment with a deep filter. In some embodiments, the deep filter includes a porous filter medium capable of holding a portion of the sample, such as cellular components and debris, and the filtration is carried out, for example, within the depth of the filter material. In some embodiments, the deep filter includes synthetic materials, non-synthetic materials, or a combination thereof. In some embodiments, the deep filter includes a substrate comprising one or more of diatomaceous earth compositions, silica compositions, cellulose fibers, polymer fibers, cohesive resins, and ash compositions. In some embodiments, at least a portion of the substrate of the deep filter includes a surface modification. In some embodiments, the surface modification is one or more of quaternary amine surface modifications, cationic surface modifications, and anionic surface modifications. In some embodiments, the deep filter is selected from the group consisting of EMPHAZE® deep filters (such as EMPHAZE® AEX deep filters), X0SP deep filters, PDD1 deep filters, ZETA PLUS® 120ZA deep filters, and ZETA PLUS® 120ZB deep filters.

[0100] In some embodiments, the deep filter comprises cellulose fibers, diatomaceous earth, and perlite. In some embodiments, the deep filter comprises two layers, each comprising a cellulose filter matrix, the cellulose filter matrix being impregnated with a filter aid comprising one or more diatomaceous earth or perlite. In some embodiments, the deep filter comprises two layers, each comprising a cellulose filter matrix, the cellulose filter matrix being impregnated with a filter aid comprising one or more diatomaceous earth or perlite, and each layer further comprising a resin binder. In some embodiments, the deep filter is a PDD1 deep filter.

[0101] In some embodiments, the deep filter comprises silica, such as a silica filter aid, and polyacrylic fibers. In some embodiments, the deep filter comprises two layers of filter media, the first layer comprising silica, such as a silica filter aid, and the second layer comprising polyacrylic fibers, such as polyacrylic fiber pulp. In some embodiments, the deep filter is a deep filter comprising synthetic materials and does not contain diatomaceous earth and / or perlite. In some embodiments, the deep filter is an X0SP deep filter.

[0102] In some embodiments, the silica filter aid is a precipitated silica filter aid. In some embodiments, the filter aid is a form of filter, such as a layer that helps perform the filtering function. In some embodiments, the silica filter aid is a silica gel filter aid. In some embodiments, the silica filter aid has about 50% silanol ionized at pH 7. In some embodiments, the silica filter aid is a silica gel filter aid, and about 50% of the silanol in the silica filter aid is ionized at pH 7. In some embodiments, the silica filter aid precipitates from silica such as SIPERNAT® (Evonik Industries AG) or silica such as Kieseigel 60 (Merck KGaA). In some embodiments, the polyacrylic fiber is a nonwoven polyacrylic fiber pulp. In some embodiments, the polyacrylic fiber is an electrospun polyacrylic nanofiber. In some embodiments, the degree of fibrillation of the polyacrylic fiber correlates with the Canadian standard filtration capacity (CSF) of about 10 mL to about 800 mL. In some embodiments, the deep filter has a pore size of about 0.05 μm to about 0.2 μm, for example, about 0.1 μm. 2 ~about 1.5m 2 For example, approximately 0.11m 2 , about 0.55m 2 , or approximately 1.1 m 2It has a surface area. In some embodiments, the deep filter does not contain diatomaceous earth and / or perlite. In some embodiments, the deep filter comprises two layers of filter media, the first layer comprising a silica filter aid having about 50% silanol ionized at pH 7, and the second layer comprising polyacrylic fiber pulp having a degree of fibrillation of polyacrylic fibers that correlates with a Canadian standard filtration capacity (CSF) of about 10 mL to about 800 mL, and the deep filter does not contain diatomaceous earth.

[0103] In some embodiments, the deep filter comprises a hydrogel Q (quaternary amine) functionalized nonwoven fabric material and a multizone microporous membrane. In some embodiments, the deep filter comprises four layers comprising the hydrogel Q functionalized nonwoven fabric material and a 9-zone microporous membrane. In some embodiments, the nonwoven fabric material comprises polypropylene. In some embodiments, the deep filter is a deep filter comprising synthetic materials and does not contain diatomaceous earth and / or perlite. In some embodiments, the deep filter is an EMPHAZE® AEX deep filter.

[0104] In some embodiments, the deep filter comprises multiple components or layers. In some embodiments, the deep filter comprises multiple layers, each comprising one or more layers comprising an anion exchange (AEX) functionalized polymer. In some embodiments, the layer comprising the AEX functionalized polymer comprises quaternary ammonium (Q), such as a Q functionalized hydrogel. In some embodiments, the layer comprising the AEX functionalized polymer comprises a quaternary ammonium (Q) functionalized polymer associated with a nonwoven article. In some embodiments, the layer comprising the AEX functionalized polymer comprises a quaternary ammonium (Q) functionalized hydrogel covalently grafted onto a microfiber polypropylene nonwoven scaffold. In some embodiments, the deep filter comprises multiple layers, each comprising a layer comprising a multizone membrane, each comprising a 9-zone membrane having a pore size of about 0.05 μm to about 0.3 μm, for example, about 0.22 μm. In some embodiments, the deep filter does not contain diatomaceous earth.

[0105] In some embodiments, the methods described herein include one or more hydrophobic interaction chromatography (HIC) steps. The HIC step is a chromatographic technique that includes treatment with an HIC medium, such as an HIC filter or HIC column. In some embodiments, the HIC medium includes a hydrophobic moiety containing, for example, methyl, ethyl, propyl, octyl, or phenyl groups. In some embodiments, the sample is applied to the HIC medium in a polar buffer. In some embodiments, polypeptides are eluted from the HIC medium using stepwise elution with aqueous buffers whose salt concentration is decreased, surfactant concentration is increased, and / or pH is adjusted.

[0106] In some embodiments, the methods described herein can reduce the enzymatic hydrolysis rate of a composition obtained from a purification platform. In some embodiments, the enzymatic hydrolysis rate represents the activity rate of one or more hydrolases, for example, one or more different hydrolases. In some embodiments, the enzymatic hydrolysis rate is a surrogate measure of the activity of one or more enzymes in the composition. In some embodiments, the enzymatic hydrolysis rate is measured by a surrogate substrate. In some embodiments, the enzymatic hydrolysis rate is evaluated by measuring the hydrolysis products of one or more hydrolases.

[0107] As used herein, the terms “comprising,” “having,” “containing,” and “including,” and their grammatical equivalents, are semantically equivalent and open-ended in that the one or more items following any one of these words do not mean an exhaustive list of such one or more items, nor do they mean that the list is limited to only the one or more items. For example, an article “comprising” components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may also contain one or more other components in addition to components A, B, and C. Accordingly, “comprises” and similar forms, and their grammatical equivalents, are intended and understood to include disclosures of embodiments of “consisting essentially of” or “consisting of.”

[0108] Where a range of values ​​is provided, unless explicitly indicated in the context, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated values ​​or intervening values ​​within that stated range, are understood to be included in this disclosure, subject to any specifically excluded limitations within that stated range. If the stated range includes one or both limits, the range excluding either or both of those included limits is also included in this disclosure.

[0109] References to values ​​or parameters “about” in this specification include (and describe) variations relating to the value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0110] When used herein and in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless the context otherwise explicitly indicates. Refining platform

[0111] In some aspects of this disclosure, a purification platform is provided that includes a deep filtration step and / or a hydrophobic interaction chromatography (HIC) step. In some embodiments, the purification platform represents a workflow for purifying a target from a sample containing the target to any degree. In some embodiments, the process workflow of the purification platform is a sequence of steps involved in the purification of a target from a sample containing the target.

[0112] For illustrative purposes of the disclosures herein, a partial sequence workflow of an exemplary purification platform 100 is shown in Figure 1A. As shown in Figure 1A, the purification platform 100 includes, but is not limited to, a sequence of steps including a capture step 105, a conditioning step 110, one or more purification steps 115 such as one or more polypeptide purification steps, a virus filtration step 120, and an ultrafiltration / diafiltration (UFDF) step 125. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more deep filtration steps. In some embodiments, the exemplary purification platform shown in Figure 1A includes a deep filtration step performed after the conditioning step 110 and before one or more purification steps. In some embodiments, the exemplary purification platform shown in Figure 1A includes a deep filtration step performed before the capture step 105. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more HIC steps. In some embodiments, the HIC step is performed after one or more purification steps, after the pH holding step of the virus filtration step, and / or after the virus filtration step. In some embodiments, the exemplary purification platform shown in Figure 1A includes one or more deep filtration steps and one or more HIC steps.

[0113] Those skilled in the art will readily understand that the purification platforms described herein lead to workflows for purifying a target from a sample containing the target, components used to carry out each step of the purification platform workflow, and components and reagents used therein. In some examples of this disclosure, descriptions of the purification platforms and their usage are provided in a modular manner. Such disclosures are not intended to limit the scope of this application. This disclosure encompasses any combination and / or arrangement of the purification platforms that are included in the disclosure of individual components and / or their steps. Deep filtration process

[0114] In some embodiments, the disclosure provides a purification platform including a deep filtration step. As described herein, the deep filtration step may be located at one or more positions within the purification platform. In some embodiments, the purification platform described herein includes one or more deep filtration steps located at any stage of the process workflow, for example, two, three, four, or five deep filtration steps. In some embodiments where the purification platform includes two or more deep filtration steps, the deep filtration steps are not performed directly and sequentially, i.e., no intervening steps of the purification platform are performed between the deep filtration steps. In some embodiments where the purification platform includes two or more deep filtration steps, the deep filtration steps are the same. In some embodiments where the purification platform includes two or more deep filtration steps, the deep filtration steps are different, including, for example, the use of different deep filters.

[0115] In some embodiments, the purification platform includes two or more deep filtration steps, where a first deep filtration step is performed before a capture step including treatment by protein A chromatography, and a second deep filtration step is performed after the capture step but before the purification step.

[0116] In some embodiments, the deep filtration process includes treatment with a deep filter. In some embodiments, the deep filter is a deep filter comprising a synthetic material. Deep filtration processes, including those involving treatment with a deep filter, are known in the art. See, for example, Yigzaw et al., Biotechnol Prog, 22, 2006, and Liu et al., mAbs, 2, 2010, which are incorporated herein by reference in their entirety. Those skilled in the art will understand, for example, the components, conditions, and reagents involved in the deep filtration process.

[0117] In some embodiments, the methods described herein include one or more deep filtration steps, each comprising treatment with a deep filter, the deep filter being selected based on the pH of the solution entering the deep filter. In some embodiments, a deep filter comprising silica and polyacrylic fibers, such as an X0SP deep filter, is selected when the solution entering the deep filter is about 5 to about 6.5. In some embodiments, a deep filter comprising silica and polyacrylic fibers, such as an X0SP deep filter, is selected when the solution entering the deep filter is about 6.5 or less, for example, 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, deep filters comprising silica and polyacrylic fibers such as X0SP deep filters are selected when the solution entering the deep filter is approximately 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, deep filters comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane such as EMPHAZE® deep filters are selected when the solution entering the deep filter is approximately 7 to approximately 8.5. In some embodiments, a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane such as an EMPHAZE® deep filter is selected when the solution entering the deep filter is about 7 or more, for example, 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 deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane such as an EMPHAZE® deep filter is selected when the solution entering the deep filter is about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.In some embodiments of the methods described herein, the method may further include the step of selecting a deep filter based on the pH of the solution to be entered into the deep filter. In some examples, it will be readily apparent to those skilled in the art that the solution to be entered into the deep filter and its properties may be based on a target such as a polypeptide (e.g., an antibody) purified using the purification platform described herein. Thus, in some embodiments, the properties of a target such as pI are used as a basis for selecting a deep filter for use with the purification platform described herein.

[0118] In some embodiments, the deep filter includes a substrate comprising one or more of the following: a diatomaceous earth composition, a silica composition, cellulose fibers, polymer fibers, a cohesive resin, synthetic particles, an ion-chargeable resin, and an ash composition. In some embodiments, the deep filter includes diatomaceous earth. In some embodiments, the deep filter includes an anion exchange medium.

[0119] In some embodiments, at least a portion of the substrate of the deep filter includes surface modification. In some embodiments, the surface modification is one or more of quaternary amine surface modification, cationic surface modification, and anionic surface modification.

[0120] In some embodiments, the deep filter is selected from the group consisting of EMPHAZE® deep filters (such as EMPHAZE® AEX deep filters), X0SP deep filters, PDD1 deep filters, ZETA PLUS® 120ZA deep filters, and ZETA PLUS® 120ZB deep filters. HIC process

[0121] In some embodiments, the disclosure provides a purification platform including HIC steps. As described herein, HIC steps can be located at any one or more positions within the purification platform. In some embodiments, the purification platform described herein includes one or more HIC steps, e.g., two, three, four, or five HIC steps, located at any stage of the process workflow. In some embodiments where the purification platform includes two or more HIC steps, the HIC steps are not performed directly and sequentially, i.e., not without some intervening steps of the purification platform performed between the HIC steps. In some embodiments where the purification platform includes two or more HIC steps, the HIC steps are the same. In some embodiments where the purification platform includes two or more HIC steps, the HIC steps are different, e.g., including the use of different HIC media.

[0122] In some embodiments, the HIC process includes treatment with an HIC medium, such as an HIC column or HIC membrane. HIC processes involving treatment with an HIC medium are known in the art. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference. Those skilled in the art will understand, for example, the components, conditions, and reagents involved in the HIC process.

[0123] 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 modifier. In some embodiments, the surface modifier is a phenyl or butyl surface modifier.

[0124] In some embodiments, the HIC process is a flow-through mode HIC process. In some embodiments, the HIC process is a binding-elution mode HIC process.

[0125] In some embodiments, the purification platform includes one or more deep filtration steps located at any stage of the process workflow, and one or more HIC steps located at any stage of the process workflow. Capture process

[0126] In some embodiments, the purification platform includes a capture step. In some embodiments, the capture step includes treatment by affinity chromatography.

[0127] For example, capture steps involving affinity chromatography are known in the art. See, for example, Liu et al. mAbs, 2, 2010, incorporated herein by reference.

[0128] In some embodiments, affinity chromatography is selected from the group consisting of protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, protein XL chromatography, FcXL chromatography, kappa chromatography, and kappa XL chromatography. In some embodiments, the capture step includes processing by protein A chromatography. In some embodiments, the capture step includes processing by FcXL chromatography.

[0129] 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.

[0130] In some embodiments, protein A chromatography is selected from the group consisting of Prose vA®, Prosep® vA Ultra, Protein A Sepharose® Fast Flow, MabSelect®, MabSelect® SuRe, Poros® A, and MabCapture®. Conditioning process

[0131] In some embodiments, the purification platform includes a conditioning step. In some embodiments, the conditioning step is performed after the capture step.

[0132] Conditioning processes, including those involving treatments for the conditioning process, are known in the art. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0133] In some embodiments, the conditioning step includes a virus inactivation step, such as a low pH maintenance step. In some embodiments, the low pH maintenance step is carried out at a pH of about 2.5 to about 4. In some embodiments, the low pH maintenance step is configured for virus inactivation. In some embodiments, the low pH maintenance step can inactivate endogenous / exogenous viruses. One or more purification steps

[0134] In some embodiments, the purification platform includes one or more purification steps. In some embodiments, one or more purification steps are performed after the capture and conditioning steps. In some embodiments, one or more purification steps include polypeptide purification steps. In some embodiments, one or more purification steps include two or more polypeptide purification steps, such as two, three, four, or five polypeptide purification steps.

[0135] Polypeptide purification processes, including those involving treatments for polypeptide purification, are known in the art. For example, see Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0136] In some embodiments, the polypeptide purification step includes treatment 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.

[0137] In some embodiments, the polypeptide purification step includes treatment by chromatography selected from the group consisting of diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), trimethylaminoethyl (TMAE), quaternary amines, quaternary aminoethyl (QAE), sulfopropyl (SP), SP-Sepharose® (cross-linked bead agarose) Fast Flow (FF), SP-Sepharose® XL, quaternary amine (Q)Sepharose® FF, mercaptoethylpyridine (MEP)-Hypercel®, Capto MMC (multimodal chromatography), Capto Adhere, Poros® XS, and Poros® 50HS.

[0138] In some embodiments, the polypeptide purification step is a bound-elution 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. Virus filtration process

[0139] In some embodiments, the purification platform includes a virus filtration step. In some embodiments, the virus filtration step is performed after one or more purification steps.

[0140] Viral filtration processes, including those involved in the processing of the viral filtration process, are known in the art. See, for example, Liu et al. mAbs, 2, 2010, incorporated herein by reference, and U.S. Patent Application Publication No. 20140309403.

[0141] In some embodiments, the virus filtration step includes treatment with a virus filter. In some embodiments, the virus filtration step includes a pH maintenance step. In some embodiments, the treatment with a virus filter is performed after the pH maintenance step. UFDF process

[0142] In some embodiments, the purification platform includes a UFDF step. In some embodiments, the UFDF step is performed after one or more purification steps and / or after a virus filtration step.

[0143] UFDF processes, including those involving the processing of the UFDF process, are known in the art. For example, see Liu et al. mAbs, 2, 2010, which is incorporated herein by reference.

[0144] In some embodiments, the UFDF process includes treatment by ultrafiltration. In some embodiments, the UFDF process is performed in tangential flow filtration (TFF) mode. In some embodiments, the UFDF process includes treatment by tangential flow filtration, such as high-performance tangential flow filtration.

[0145] As described above, the purification platform disclosed in this application may include any combination and arrangement of purification steps, including those described herein. For example, in some embodiments, the purification platform includes a capture step and a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0146] In some embodiments, the purification platform includes, in sequence, a capture step and a conditioning step, the purification platform further including a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0147] In some embodiments, the purification platform comprises, in sequence, a capture step, a conditioning step, and one or more purification steps, and the purification platform further comprises a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed between or after one or more purification steps. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further comprises a HIC step, such as a HIC step performed after one or more purification steps.

[0148] 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, and the purification platform further comprises a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after any of one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the deep filtration step is performed after the viral filtration step. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, which is selected from one or more HIC steps performed after and / or during one or more purification steps, such as a viral filtration step, a pH holding step of a viral filtration step, etc.

[0149] In some embodiments, the purification platform comprises, in sequence, a capture step, a conditioning step, one or more purification steps, and a UFDF step, and the purification platform further comprises a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during any of one or more purification steps or after one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further comprises an HIC step, such as one or more HIC steps selected from one or more HIC steps performed after one or more purification steps and / or after the UFDF step.

[0150] In some embodiments, the purification platform includes a capture step, a conditioning step, one or more purification steps, a viral filtration step, and a UFDF step, and the purification platform further includes a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the deep filtration step is performed after the viral filtration step and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further includes a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, which is selected from one or more HIC steps performed after one or more purification steps and / or after or during a viral filtration step, such as after a pH holding step in a viral filtration step, and / or after a UFDF step.

[0151] In some embodiments, the purification platform includes a capture step and a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0152] In some embodiments, the purification platform includes, in sequence, a capture step and a conditioning step, the purification platform further including a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step, performed after the capture step.

[0153] In some embodiments, the purification platform comprises, in sequence, a capture step, a conditioning step, and one or more purification steps, and the purification platform further comprises 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during or after any of the one or more purification steps. In some embodiments, the purification platform further comprises a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further comprises a deep filtration step, such as a deep filtration step performed after the capture step.

[0154] In some embodiments, the purification platform comprises a conditioning step, one or more purification steps, and a virus filtration step in sequence, and the purification platform further comprises 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during any of one or more purification steps or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before 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 an HIC step performed after one or more purification steps. In some embodiments, the purification platform further comprises a deep filtration step, such as a deep filtration step performed after the capture step.

[0155] In some embodiments, the purification platform includes a capture step, a conditioning step, one or more purification steps, and a UFDF step, and 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during any of one or more purification steps or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0156] In some embodiments, the purification platform includes a capture step, a conditioning step, one or more purification steps, a viral filtration step, and a UFDF step, and 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the HIC step is performed after the viral filtration 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 one or more purification steps. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step. Sample, its components, and composition obtained from the purification platform.

[0157] In some embodiments, the purification platform described herein is useful for purifying a target from a sample containing the target to any degree.

[0158] In some embodiments, the sample is a host cell sample. In some embodiments, the sample is a host cell culture medium (HCCF). In some embodiments, the sample contains a portion of the host cell culture medium. In some embodiments, the sample is derived from the host cell culture medium. In some embodiments, the sample contains host cells. In some embodiments, the sample contains host cell components such as host cell debris. In some embodiments, the host cells are bacterial cells. In some embodiments, the host cells are insect cells. In some embodiments, the host cells are mammalian cells. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the host cells are Escherichia coli (E. coli) cells.

[0159] In some embodiments, the sample is treated, such as being subjected to a processing step performed before being subjected to the purification platform described herein. In some embodiments, the sample comprises a surfactant. In some embodiments, the sample comprises a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0160] In some embodiments, the sample includes a target. In some embodiments, the target includes 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 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-α-beta 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 moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0161] In some embodiments, the sample comprises one or more host cell proteins. In some embodiments, the host cell protein is a hydrolase. In some embodiments, the hydrolase is a lipase, esterase, thioesterase, phospholipase, or ceramidase. In some embodiments, the hydrolase is a polyenzyme protein. In some embodiments, the polyenzyme protein is a fatty acid synthase. In some embodiments, the fatty acid synthase comprises a thioesterase subunit.

[0162] The purification platforms described herein may, in some cases, include a number of purification steps. In some embodiments, the term “composition” is used herein to describe any input (except 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 describing the final output of the purification platform.

[0163] In some embodiments, the composition includes a surfactant. In some embodiments, the composition includes a polysorbate. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0164] 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 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-α-beta 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 moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0165] 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, esterase, thioesterase, phospholipase, or ceramidase. Additional steps

[0166] In some embodiments, this disclosure provides additional steps involved in or related to the purification platform described herein. Additional steps involved in or related to the purification platform and methods for performing such steps are known. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein in its entirety by reference.

[0167] In some embodiments, the purification platform further includes a sample preparation step, such as a sample preparation step. In some embodiments, the purification platform further includes a clarification step, for example, for clarifying HCCF. In some embodiments, the purification platform further includes a host cell and host cell residue removal step, for example, for removing host cells and host cell residue from the sample and / or composition obtained from the purification platform. In some embodiments, the purification platform further includes a centrifugation step. In some embodiments, the purification platform further includes a sterile filtration step. In some embodiments, the purification platform further includes a tangential flow microfiltration step. In some embodiments, the purification platform further includes a flocculation / precipitation step. How to use a purification platform

[0168] In some embodiments, this disclosure describes a method using the purification platform described herein. In some embodiments, the method includes subjecting a sample containing a target to the purification platform described herein.

[0169] In some embodiments, a method is provided herein for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform described herein, comprising one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction in the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps is at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the enzymatic hydrolysis rate is the enzymatic polysorbate hydrolysis rate.

[0170] In some embodiments, a method is provided herein for reducing the level of one or more hydrolases in a composition obtained from a purification platform described herein that includes one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby reducing the level of hydrolases in the composition compared to the purification of a sample using the same purification platform that does not include one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction in the level of one or more hydrolases in the composition compared to the purification of a sample using the same purification platform that does not include one or more deep filtration steps and / or one or more HIC steps is at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, one or more hydrolases can hydrolyze polysorbate.

[0171] In some embodiments, a method is provided herein for reducing the degradation of polysorbate in a composition obtained from a purification platform described herein that includes one or more deep filtration steps and / or one or more HIC steps, comprising subjecting a sample to the purification platform, thereby reducing the degradation of polysorbate in the composition compared to the purification of a sample using the same purification platform that does not include one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative reduction of the degradation of polysorbate in the composition compared to the purification of a sample using the same purification platform that does not include one or more deep filtration steps and / or one or more HIC steps is at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0172] In some embodiments, methods are provided herein for extending the shelf life of a composition obtained from a purification platform described herein, which includes one or more deep filtration steps and / or one or more HIC steps, comprising subjecting a sample to the purification platform, thereby extending the shelf life of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the relative increase in shelf life of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps is at least about one week, for example, at least about two weeks, one month, two months, three months, four months, five months, six months, nine months, twelve months, eighteen months, twenty-four months, or more than twenty-four months. In some embodiments, the shelf life of the composition is at least about one week, for example, at least about two weeks, one month, two months, three months, four months, five months, six months, nine months, twelve months, eighteen months, twenty-four months, thirty months, thirty-six months, forty-two months, forty-eight months, or more than fourty-eight months, compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the shelf life of the composition is more than six months, more than nine months, more than twelve months, more than eighteen months, more than twenty-four months, more than thirty months, more than thirty-six months, more than forty-two months, more than forty-eight months, or more than fourty-eight months.

[0173] In some embodiments, a method is provided herein for producing a composition with less polysorbate degradation, the composition obtained from a purification platform described herein which includes one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby producing a composition with less polysorbate degradation compared to the purification of a sample using the same purification platform which does not include one or more deep filtration steps and / or one or more HIC steps.

[0174] In some embodiments, the present invention provides a method for reducing the aggregation of a target in a composition obtained from a purification platform described herein, which includes one or more deep filtration steps and / or one or more HIC steps, the method comprising subjecting a sample to the purification platform, thereby reducing the aggregation of a target in the composition compared to the purification of a sample using the same purification platform, which does not include one or more deep filtration steps and / or one or more HIC steps.

[0175] As described herein, one or more attributes of a composition obtained by subjecting a sample to a purification platform including one or more deep filtration steps and / or one or more HIC steps are compared to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. Those skilled in the art will understand that, in some cases, such comparisons must be carried out under appropriate conditions to enable a meaningful comparison. For example, when comparing a composition obtained from a purification platform including one or more deep filtration steps and / or one or more HIC steps to the purification of a sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps, temporal factors that may affect the reading of the enzyme hydrolysis activity rate must be taken into consideration. Further relevant factors to be considered for comparison between a composition and a reference include experimental conditions, assays used, temperature conditions, pH, timing, sample, buffer, and sample source.

[0176] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which includes a capture step and a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0177] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which sequentially includes a capture step and a conditioning step, the purification platform further including a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step. In some embodiments, the purification platform further includes a second deep filtration step. In some embodiments, the purification platform further includes a HIC step.

[0178] In some embodiments, the method comprises subjecting a sample containing a target to a purification platform comprising a capture step, a conditioning step, and one or more purification steps in this order, the purification platform further comprising a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after any of the one or more purification steps. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further comprises a HIC step, such as a HIC step performed after one or more purification steps.

[0179] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence, a capture step, a conditioning step, one or more purification steps, and a viral filtration step, the purification platform further comprising a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after any of one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the deep filtration step is performed after the viral filtration step. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, which is selected from one or more HIC steps performed after and / or during one or more purification steps, such as a viral filtration step, a pH holding step of a viral filtration step, etc.

[0180] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence, a capture step, a conditioning step, one or more purification steps, and a UFDF step, the purification platform further comprising a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after any of one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, which is selected from one or more HIC steps performed after one or more purification steps and / or after a UFDF step.

[0181] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising a conditioning step, one or more purification steps, a viral filtration step, and a UFDF step, the purification platform further comprising a deep filtration step. In some embodiments, the deep filtration step is performed before the capture step. In some embodiments, the deep filtration step is performed after the capture step and before the conditioning step. In some embodiments, the deep filtration step is performed after the conditioning step and before one or more purification steps. In some embodiments, if there are two or more purification steps, the deep filtration step is performed during or after one or more purification steps. In some embodiments, the deep filtration step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the deep filtration step is performed after the viral filtration step and before the UFDF step. In some embodiments, the deep filtration step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second deep filtration step, such as a deep filtration step performed before the capture step. In some embodiments, the purification platform further includes a HIC step, which is selected from one or more HIC steps performed after or between one or more purification steps and / or after a pH holding step of a viral filtration step, a viral filtration step and / or a UFDF step.

[0182] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which includes a capture step and a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0183] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which sequentially includes a capture step and a conditioning step, the purification platform further including a HIC step. In some embodiments, the HIC step is performed before the capture step. In some embodiments, the HIC step is performed after the capture step and before the conditioning step. In some embodiments, the HIC step is performed after the conditioning step. In some embodiments, the purification platform further includes a second HIC step. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0184] In some embodiments, the method comprises subjecting a sample containing a target to a purification platform comprising a capture step, a conditioning step, and one or more purification steps in this order, the purification platform further comprising 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during or after any of the one or more purification steps. In some embodiments, the purification platform further comprises a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further comprises a deep filtration step, such as a deep filtration step performed after the capture step.

[0185] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which sequentially includes a capture step, a conditioning step, one or more purification steps, and a viral filtration step, the purification platform further including 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during any of one or more purification steps or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the HIC step is performed after the viral filtration step. In some embodiments, the purification platform further includes a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0186] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in sequence, a capture step, a conditioning step, one or more purification steps, and a UFDF step, the purification platform further comprising 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during any of one or more purification steps or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before the UFDF step. In some embodiments, the HIC step is performed after the UFDF step. In some embodiments, the purification platform further comprises a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further comprises a deep filtration step, such as a deep filtration step performed after the capture step.

[0187] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising a capture step, a conditioning step, one or more purification steps, a viral filtration step, and a UFDF step, the purification platform further comprising an 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 one or more purification steps. In some embodiments, if there are two or more purification steps, the HIC step is performed during or after one or more purification steps. In some embodiments, the HIC step is performed after one or more purification steps and before the viral filtration step. In some embodiments, the HIC step is performed after the viral filtration 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 comprises a second HIC step, such as an HIC step performed after one or more purification steps. In some embodiments, the purification platform further includes a deep filtration step, such as a deep filtration step performed after the capture step.

[0188] For illustrative purposes of the disclosures herein, Figure 1B shows a sequence workflow of options available for an exemplary embodiment of the purification platform 200. As shown in Figure 1B, the purification platform includes: a protein A chromatography step 210; a further chromatography step selected from HIC 225, cation exchange chromatography 225, or multimodal chromatography 230; and one or more deep filtration steps selected from EMPHAZE® deep filtration step 205 performed on HCCF prior to the protein A chromatography step, X0SP deep filtration step 215 performed on a protein A pool, or EMPHAZE® deep filtration step 220 performed on a protein A pool.

[0189] As shown in Figure 1B, in some embodiments, the purification platform includes an EMPHAZE® deep filtration step 205 performed on HCCF before subjecting the EMPHAZE® deep filtration pool to protein A chromatography 210. In such embodiments, the protein A pool is subjected to either an X0SP deep filtration step 215 or an EMPHAZE® deep filtration step 220 before the downstream chromatography step. In some embodiments where the purification platform includes an X0SP deep filtration step 215, the protein A pool is conditioned before the X0SP deep filtration step 215 by adjusting the pH of the protein A pool to about 5 to about 6.5. In some embodiments where the purification platform includes an X0SP deep filtration step 215, the X0SP deep filtration pool is further subjected to HIC (e.g., phenylSEPHAROSE® high-speed flow) or cation exchange chromatography (e.g., POROS® 50HS). In some embodiments of the purification platform, which includes an EMPHAZE® deep filtration step 220, the Protein A pool is conditioned prior to the EMPHAZE® deep filtration step 220 by adjusting the pH of the Protein A pool to about 7 to about 8.5. In some embodiments of the purification platform, which includes an EMPHAZE® deep filtration step 220, the EMPHAZE® deep filtration pool is further subjected to multimodal chromatography (e.g., CaptoAdhere).

[0190] As shown in Figure 1B, in some embodiments, the purification platform does not include an EMPHAZE® deep filtration step 205 performed on the HCCF. In such embodiments, the HCCF is subjected to protein A chromatography 210, and the protein A pool is subjected to either an X0SP deep filtration step 215 or an EMPHAZE® deep filtration step 220 before the downstream chromatography step. In some embodiments where the purification platform includes an X0SP deep filtration step 215, the protein A pool is conditioned before the X0SP deep filtration step 215 by adjusting the pH of the protein A pool to about 5 to about 6.5. In some embodiments where the purification platform includes an X0SP deep filtration step 215, the X0SP deep filtration pool is further subjected to HIC (e.g., Phenyl Sepharose® High-Speed ​​Flow) or cation exchange chromatography (e.g., POROS® 50HS). In some embodiments of the purification platform, which includes an EMPHAZE® deep filtration step 220, the Protein A pool is conditioned prior to the EMPHAZE® deep filtration step 220 by adjusting the pH of the Protein A pool to about 7 to about 8.5. In some embodiments of the purification platform, which includes an EMPHAZE® deep filtration step 220, the EMPHAZE® deep filtration pool is further subjected to multimodal chromatography (e.g., CaptoAdhere).

[0191] In some embodiments, the method involves subjecting a sample containing a target to a purification platform comprising, in this order: (a) a deep filtration step including treatment with a deep filter comprising a hydrogel Q-functionalized nonwoven medium and a multizone microporous membrane; (b) a capture step including treatment with protein A chromatography; and (c) a purification step, wherein the purification step includes treatment with chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography. In some embodiments, the deep filter is an EMPHAZE® deep filter. In some embodiments, the HIC is phenyl SEPHAROSE® high-speed flow chromatography. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the multimodal chromatography is Capto Adhere.

[0192] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in this order: (a) a first deep filtration step including treatment with a first deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane; (b) a capture step including treatment by protein A chromatography; (c) a second deep filtration step including treatment with a second deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane; and (d) a purification step including treatment by multimodal chromatography. In some embodiments, the first and second deep filters are EMPHAZE® deep filters. In some embodiments, the multimodal chromatography is Capto Adhere. In some embodiments, the second deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane is selected when the solution entering the deep filter is about 7 to about 8.5.

[0193] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in this order: (a) a capture step including treatment by protein A chromatography; (b) a deep filtration step including treatment by a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane; and (c) a purification step including treatment by multimodal chromatography. In some embodiments, the deep filter is an EMPHAZE® deep filter. In some embodiments, the multimodal chromatography is Capto Adhere. In some embodiments, the deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane is selected when the volume of solution entering the deep filter is about 7 to about 8.5.

[0194] In some embodiments, the method includes subjecting a sample containing a target to a purification platform comprising, in this order: (a) a first deep filtration step including treatment with a first deep filter; (b) a capture step including treatment by protein A chromatography; and (c) a second deep filtration step including treatment with a second deep filter. In some embodiments, the first deep filter comprises a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane. In some embodiments, the first deep filter is an EMPHAZE® deep filter. In some embodiments, the first deep filter comprises an inorganic filter aid, cellulose, and a resin system. In some embodiments, the first deep filter is a 120 ZB deep filter. In some embodiments, the second deep filter comprises silica and polyacrylic fibers. In some embodiments, the second deep filter is an X0SP deep filter. In some embodiments, the method further includes a conditioning step. In some embodiments, the method further includes one or more purification steps.

[0195] In some embodiments, the method involves subjecting a sample containing a target to a purification platform, which comprises, in this order: (a) a first deep filtration step including treatment with a first deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane; (b) a capture step including treatment by protein A chromatography; (c) a second deep filtration step including treatment with a second deep filter comprising silica and polyacrylic fibers; and (d) a purification step including treatment by cation exchange chromatography. In some embodiments, the first deep filter is an EMPHAZE® deep filter. In some embodiments, the second deep filter is an X0SP deep filter. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the second deep filter comprising silica and polyacrylic fibers is selected when the solution entering the deep filter is about 5 to about 6.5.

[0196] In some embodiments, the method involves subjecting a sample containing a target to a purification platform, which comprises, in this order: (a) a first deep filtration step including treatment with a first deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane; (b) a capture step including treatment with protein A chromatography; (c) a second deep filtration step including treatment with a second deep filter comprising silica and polyacrylic fibers; and (d) a purification step including treatment with HIC. In some embodiments, the first deep filter is an EMPHAZE® deep filter. In some embodiments, the second deep filter is an X0SP deep filter. In some embodiments, the HIC is phenylSEPHAROSE® high-speed flow chromatography. In some embodiments, the second deep filter comprising silica and polyacrylic fibers is selected when the solution entering the deep filter is about 5 to about 6.5.

[0197] In some embodiments, the method includes subjecting a sample containing a target to a purification platform which includes, in this order, a capture step including treatment by protein A chromatography and a deep filtration step including treatment by a deep filter. In some embodiments, the deep filter includes silica and polyacrylic fibers. In some embodiments, the deep filter is an X0SP deep filter. In some embodiments, the method further includes a conditioning step. In some embodiments, the method further includes one or more purification steps.

[0198] In some embodiments, the method involves subjecting a sample containing a target to a purification platform that includes, in this order: (a) a capture step including treatment by protein A chromatography; (b) a deep filtration step including treatment by a deep filter containing silica and polyacrylic fibers; and (c) a purification step including treatment by cation exchange chromatography. In some embodiments, the deep filter is an X0SP deep filter. In some embodiments, the cation exchange chromatography is POROS® 50HS. In some embodiments, the deep filter containing silica and polyacrylic fibers is selected when the amount of solution entering the deep filter is about 5 to about 6.5.

[0199] In some embodiments, the method involves subjecting a sample containing a target to a purification platform comprising, in this order: (a) a capture step including treatment by protein A chromatography; (b) a deep filtration step including treatment by a deep filter containing silica and polyacrylic fibers; and (c) a purification step including treatment by HIC. In some embodiments, the deep filter is an X0SP deep filter. In some embodiments, the HIC is phenyl Sepharose® high-performance flow chromatography. In some embodiments, the deep filter containing silica and polyacrylic fibers is selected when the volume of solution entering the deep filter is about 5 to about 6.5. Additional method steps

[0200] In some embodiments, the method described herein further includes additional method steps. In some embodiments, the method further includes a cell culture step. In some embodiments, the method further includes a formulation step, such as processing the composition to form a pharmaceutically acceptable composition or a precursor thereof.

[0201] In some embodiments, the method further includes determining the enzymatic hydrolysis activity rate of the composition. In some embodiments, the method further includes performing a lipase activity assay on the composition obtained from the purification platform described herein. In some embodiments, the lipase activity assay includes the step of measuring the lipase activity of one or more hydrolases by monitoring the conversion of a substrate (e.g., a non-fluorescent substrate) to a detectable product (e.g., a fluorescent product) of the hydrolase. In some embodiments, the substrate includes an ester bond. In some embodiments, the method further includes determining the product of one or more hydrolases, as described, for example, in International Publication No. 2018035025, which is incorporated herein in its entirety by reference. In some embodiments, the method further includes determining the level of free fatty acids (FFA) in the composition obtained from the purification platform described herein by performing a fatty acid mass spectrometry (FAMS) assay. In some embodiments, the method further includes determining the level of one or more hydrolases in the composition. In some embodiments, the method further includes determining the shelf life of the composition. In some embodiments, the method further includes determining the level of target aggregates in the composition. Pharmaceutical composition

[0202] In some embodiments, this disclosure provides pharmaceutical compositions 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.

[0203] In some embodiments, the pharmaceutical composition comprises an antibody moiety. In some embodiments, the pharmaceutical composition comprises an antibody moiety and a polysorbate. In some embodiments, the pharmaceutical composition comprises an antibody moiety, a polysorbate, and host cell impurities such as host cell proteins, e.g., hydrolytic enzymes.

[0204] In some embodiments, the pharmaceutical composition includes polysorbate. In some embodiments, the pharmaceutical composition is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0205] In some embodiments, the pharmaceutical composition exhibits a reduced enzymatic hydrolysis activity rate compared to a composition obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0206] In some embodiments, the pharmaceutical composition exhibits a reduced level of one or more hydrolytic enzymes compared to a composition obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0207] In some embodiments, the pharmaceutical composition exhibits reduced polysorbate degradation compared to compositions obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0208] In some embodiments, the pharmaceutical composition has an increased shelf life compared to a composition obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0209] In some embodiments, the pharmaceutical composition contains less degraded polysorbate compared to a composition obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0210] In some embodiments, the pharmaceutical composition exhibits reduced target aggregation compared to compositions obtained from the purification of the same sample using the same purification platform without one or more deep filtration steps and / or one or more HIC steps. Formulated antibody partial composition

[0211] In some embodiments, this disclosure provides formulated antibody partial compositions obtained from the purification platform described herein. In some embodiments, the formulated antibody partial compositions are obtained from the method described herein.

[0212] In some embodiments, the formulated antibody partial composition comprises an antibody portion. In some embodiments, the formulated antibody partial composition comprises an antibody portion and a polysorbate. In some embodiments, the formulated antibody partial composition comprises an antibody portion, a polysorbate, and host cell impurities (such as host cell proteins, e.g., hydrolytic enzymes).

[0213] In some embodiments, the formulated antibody partial compositions described herein have increased shelf life compared to a reference formulated antibody partial composition obtained from the same purification platform without, for example, one or more deep filtration steps and / or one or more HIC steps. In some embodiments, shelf life is assessed (e.g., by measurement) by aggregation of the antibody portion of the formulated antibody partial composition. In some embodiments, shelf life is assessed by measurement or the like by preservation of one or more functionalities of the antibody portion of the formulated antibody partial composition. In some embodiments, shelf life is assessed by measurement or the like by the activity of the antibody portion of the formulated antibody partial composition, for example, by binding activity.

[0214] In some embodiments, the formulated antibody partial composition comprising an antibody moiety and polysorbate exhibits a reduced polysorbate hydrolysis activity rate, and the shelf life of the composition is any of the following: more than about 12 months, for example, more than about 13 months, more than about 14 months, more than about 15 months, more than about 16 months, more than about 17 months, more than about 18 months, more than about 19 months, more than about 20 months, more than about 21 months, more than about 22 months, more than about 23 months, more than about 24 months, more than about 25 months, more than about 26 months, more than about 27 months, more than about 28 months, more than about 29 months, more than about 30 months, more than about 31 months, more than about 32 months, more than about 33 months, more than about 34 months, more than about 35 months, or more than about 36 months. In some embodiments, the formulated antibody partial composition with reduced polysorbate hydrolysis activity rate is compared to a reference, for example, a formulated antibody partial composition obtained from the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the reduced polysorbate hydrolysis rate is the reduced relative polysorbate hydrolysis rate.

[0215] In some embodiments, the formulated antibody partial composition comprising an antibody moiety and polysorbate exhibits a reduced polysorbate hydrolysis rate, extending the shelf life of the composition compared to the shelf life indicated in documentation submitted to the health authorities relating to the formulated antibody partial composition, by at least about two months, for example, at least about three, four, five, six, seven, eight, nine, ten, eleven, or twelve months compared to the shelf life indicated in said documentation. In some embodiments, the formulated antibody partial composition with reduced polysorbate hydrolysis rate is compared to a reference, for example, a formulated antibody partial composition obtained from the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the reduced polysorbate hydrolysis rate is a reduced relative polysorbate hydrolysis rate.

[0216] In some embodiments, the formulated antibody partial composition comprising an antibody moiety and polysorbate exhibits reduced polysorbate degradation, where the degradation is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, compared to the degradation shown in documentation submitted to health authorities relating to the formulated antibody partial composition. In some embodiments, the formulated antibody partial composition with reduced polysorbate degradation is compared to a reference, for example, a formulated antibody partial composition obtained from the same purification platform without one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the reduction in polysorbate degradation is a reduction in relative polysorbate degradation.

[0217] In some embodiments, the polysorbate hydrolysis activity rate of the formulated antibody partial composition is reduced by at least about 5% compared to the reference, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0218] In some embodiments, the formulated antibody partial composition comprises an antibody partial and a polysorbate, the polysorbate being degraded at a rate of approximately 50% or less per year during storage of the liquid composition, for example, approximately 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.

[0219] In some embodiments, the formulated antibody partial compositions described herein exhibit reduced aggregate formation for at least about 6 months, for example, at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, compared to a formulated antibody partial composition obtained from the same purification platform without, for example, one or more deep filtration steps and / or one or more HIC steps. In some embodiments, the formulated antibody partial compositions described herein have at least about 20% less aggregate formation compared to a reference for at least about 6 months, for example at least about 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months, for at least about 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, or 100% less aggregate formation compared to a reference, where the reference is a formulated antibody partial composition obtained from the same purification platform that does not include one or more deep filtration steps and / or one or more HIC steps. Methods for evaluating aggregate formation, such as measurement, are known in the art and include, for example, visual inspection, dynamic light scattering, static light scattering, and optical density measurement.

[0220] In some embodiments, the formulated antibody partial compositions described herein maintain at least about 50% of the antibody partial activity, for example at least about 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, compared to a reference, for at least about 6 months, for example at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, where the reference is a formulated antibody partial composition obtained from the same purification platform without one or more deep filtration steps and / or one or more HIC steps.

[0221] In some embodiments, the antibody portion is a monoclonal antibody.

[0222] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0223] In some embodiments, the antibody is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α-beta 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.

[0224] In some embodiments, the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0225] In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0226] Further embodiments reported herein are formulated antibody compositions exhibiting low polysorbate degradation during storage. One embodiment of the present invention is a formulated antibody composition comprising an antibody / protein and a polysorbate, wherein the polysorbate is degraded by 20% or less per year during the storage period of the formulated antibody composition (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). In one embodiment, the polysorbate is degraded by 10% or less per year during storage of the liquid composition.

[0227] Another embodiment is a formulated antibody composition comprising an antibody and a polysorbate, wherein after one year the polysorbate is present in the composition at a concentration of at least 80% of the initial concentration (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, and at least 99% in one embodiment), where the initial concentration is the concentration of the antibody in the liquid composition at the time of formulation or at the start of storage. Exemplary Embodiments

[0228] Embodiment 1. A method for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, comprising subjecting a sample to a purification platform including (a) a capture step and (b) a deep filtration step, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0229] Embodiment 2. The method according to Embodiment 1, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

[0230] Embodiment 3. The method according to Embodiment 1 or 2, wherein the relative decrease in the enzymatic hydrolysis activity rate of the composition is at least about 20% compared to the purification of a sample using the same purification platform without a deep filtration step.

[0231] Embodiment 4. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising (a) a capture step and (b) a deep filtration step, thereby reducing the level of hydrolytic enzymes in the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0232] Embodiment 5. The method according to Embodiment 4, wherein one or more hydrolytic enzymes can hydrolyze the polysorbate.

[0233] Embodiment 6. The method according to Embodiment 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 the purification of a sample using the same purification platform without a deep filtration step.

[0234] Embodiment 7. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising (a) a capture step and (b) a deep filtration step, thereby reducing the degradation of polysorbate in the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0235] Embodiment 8. The method according to Embodiment 7, wherein the relative reduction in the degradation of polysorbate in the composition is at least about 5% compared to the purification of a sample using the same purification platform without a deep filtration step.

[0236] Embodiment 9. The method according to any one of Embodiments 1 to 8, wherein the purification platform is for the purification of a target from a sample, and the sample contains a target and one or more host cell impurities.

[0237] Embodiment 10. The method according to Embodiment 9, wherein the target comprises a polypeptide.

[0238] Embodiment 11. The method according to Embodiment 9 or 10, wherein the host cell impurity is a host cell protein.

[0239] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the deep filtration step is performed before the capture step, or the deep filtration step is performed after the capture step.

[0240] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein the deep filtration step includes processing with a deep filter.

[0241] Embodiment 14. The method according to Embodiment 13, wherein the deep filter comprises a substrate containing one or more diatomaceous earth compositions, silica compositions, cellulose fibers, polymer fibers, agglomerating resins, and ash compositions.

[0242] Embodiment 15. The method according to Embodiment 14, wherein at least a portion of the substrate of the deep filter includes surface modification.

[0243] Embodiment 16. The method according to Embodiment 15, wherein the surface modification is one or more of quaternary amine surface modification, cationic surface modification, and anionic surface modification.

[0244] Embodiment 17. The method according to any one of Embodiments 14 to 16, wherein the deep filter is selected from the group consisting of EMPHAZE® deep filter, PDD1 deep filter, ZETA PLUS® 120ZA deep filter, and ZETA PLUS® 120ZB deep filter.

[0245] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the capture step includes processing by affinity chromatography.

[0246] Embodiment 19. The method according to Embodiment 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.

[0247] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the purification platform further includes a virus inactivation step, the virus inactivation step being performed after the capture step.

[0248] Embodiment 21. The method according to Embodiment 20, wherein the deep filtration step is performed after the virus inactivation step.

[0249] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the purification platform further comprises another deep filtration step performed prior to the capture step.

[0250] Embodiment 23. The method according to any one of Embodiments 1 to 22, wherein the purification platform further comprises one or more purification steps, the one or more purification steps being carried out after a capture step, a deep filtration step, and, if present, a virus inactivation step.

[0251] Embodiment 24. The method according to Embodiment 23, wherein one or more purification steps include a polypeptide purification step.

[0252] Embodiment 25. The method according to Embodiment 23 or 24, wherein the purification platform further comprises another deep filtration step performed before, during, or after one or more purification steps.

[0253] Embodiment 26. The method according to any one of Embodiments 1 to 25, wherein the purification platform further includes an ultrafiltration / diafiltration (UFDF) step, and the UFDF step is performed after one or more purification steps.

[0254] Embodiment 27. The method according to Embodiment 26, wherein the purification platform further includes another deep filtration step performed before or after the UFDF step.

[0255] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step.

[0256] Embodiment 29. The method according to Embodiment 28, wherein, if an HIC purification step is present, it is performed before, during, or after one or more purification steps.

[0257] The method according to Embodiment 28, wherein the HIC purification step is carried out after one or more purification steps and, if present, before the UFDF step.

[0258] The method according to Embodiment 26 or 27, wherein the purification platform further comprises a pH maintenance step, and the pH maintenance step is carried out after one or more purification steps and, if present, before the UFDF step.

[0259] The method according to Embodiment 31, wherein the purification platform further comprises a virus filtration step, and the virus filtration step is carried out after the pH maintenance step and, if present, before the UFDF step.

[0260] The method according to Embodiment 32, wherein the virus filtration step comprises treatment with a virus filter.

[0261] The method according to Embodiment 28, wherein the HIC purification step comprises treatment with a HIC filter.

[0262] The method according to any one of Embodiments 23 to 34, wherein each of the one or more purification steps independently comprises treatment 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.

[0263] The method according to any one of Embodiments 23 to 35, wherein each of the one or more purification steps independently comprises treatment by chromatography selected from the group consisting of DEAE, DMAE, TMAE, QAE, SPSFF, SPXL, QSFF, MEP-Hypercel™, Capto MMC, and Capto Adhere.

[0264] Embodiment 37. A method for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order: (a) a capture step including treatment by affinity chromatography; (b) a virus 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 deep filtration step carried out at one or more of the following locations: (i) before the capture step; (ii) after the capture step and before the virus inactivation step; (iii) after the virus inactivation step and before the second polypeptide purification step; (iv) after the second polypeptide purification step and before the third polypeptide purification step; or (v) after the third polypeptide purification step and before the ultrafiltration / diafiltration (UFDF) step, thereby reducing the enzymatic hydrolysis rate of the composition compared to the purification of a sample using the same purification platform without the deep filtration step.

[0265] Embodiment 38. The method according to Embodiment 37, wherein the purification platform further includes a pH holding step and a virus filtration step, in this order, performed after the third polypeptide purification step and before the UFDF step.

[0266] Embodiment 39. The method according to Embodiment 38, wherein the virus filtration step includes treatment with a virus filter.

[0267] Embodiment 40. The method according to any one of Embodiments 37 to 39, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step performed in one or more of the following: (i) after the third polypeptide purification step and before the pH holding step, (ii) after the pH holding step and before the viral filtration step, or (iii) after the viral filtration step and before the UFDF step.

[0268] Embodiment 41. The method according to any one of Embodiments 1 to 40, further comprising determining the enzymatic hydrolysis activity rate of the composition.

[0269] Embodiment 42. The method according to any one of Embodiments 1 to 41, further comprising determining the level of one or more hydrolytic enzymes in the composition.

[0270] Embodiment 43. The method according to any one of Embodiments 1 to 42, wherein the composition comprises a polysorbate.

[0271] Embodiment 44. The method according to Embodiment 43, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0272] Embodiment 45. The method according to any one of Embodiments 1 to 44, further comprising a sample processing step.

[0273] Embodiment 46. The method according to any one of Embodiments 1 to 45, wherein the sample is a cell culture sample or derived from a cell culture sample.

[0274] Embodiment 47. The method according to Embodiment 46, wherein the cell culture sample comprises host cells, and the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells.

[0275] Embodiment 48. The method according to any one of Embodiments 1 to 47, wherein the sample comprises a host cell or a component derived therefrom.

[0276] Embodiment 49. The method according to any one of Embodiments 1 to 48, wherein the sample comprises one or more host cell proteins, and one of the one or more host cell proteins is a hydrolase.

[0277] Embodiment 50. The method according to Embodiment 49, wherein the hydrolytic enzyme is lipase, esterase, thioesterase, phospholipase, or ceramidase.

[0278] Embodiment 51. The method according to any one of Embodiments 1 to 50, wherein the sample comprises a target, and the target is an antibody portion.

[0279] Embodiment 52. The method according to Embodiment 51, wherein the antibody portion is a monoclonal antibody.

[0280] Embodiment 53. The method according to Embodiment 51 or 52, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0281] Embodiment 54. The method according to any one of Embodiments 51 to 53, wherein the antibody portion is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α-beta 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.

[0282] Embodiment 55. The method according to any one of Embodiments 51 to 54, wherein the antibody portion is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0283] Embodiment 56. A pharmaceutical composition obtained by the method described in any one of Embodiments 1 to 55.

[0284] Embodiment 57. A formulated antibody partial composition comprising an antibody moiety and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate and a shelf life of more than 24 months.

[0285] Embodiment 58. A formulated antibody portion composition comprising an antibody portion and polysorbate, wherein the composition has a reduced rate of polysorbate hydrolysis, and the shelf life of the composition is extended as compared to the shelf life shown in the document submitted to the health authority related to the formulated antibody portion composition, and the shelf life is extended by at least 6 months as compared to the shelf life shown in the document, the formulated antibody portion composition.

[0286] Embodiment 59. A formulated antibody portion composition comprising an antibody portion, wherein the degradation of polysorbate in the formulated antibody portion composition is reduced, and the degradation is reduced by at least about 20% as compared to the degradation shown in the document submitted to the health authority related to the formulated antibody portion composition, the formulated antibody portion composition.

[0287] Embodiment 60. A formulated antibody portion composition comprising an antibody portion and polysorbate, wherein the polysorbate is degraded by 20% or less per year during storage of the liquid composition, the formulated antibody portion composition.

[0288] Embodiment 61. The formulated antibody portion composition according to any one of Embodiments 57 to 60, wherein the antibody portion is a monoclonal antibody.

[0289] Embodiment 62. The formulated antibody portion composition according to any one of Embodiments 57 to 61, wherein the antibody portion is a human antibody, a humanized antibody or a chimeric antibody.

[0290] Embodiment 63. A formulated antibody partial composition according to any one of Embodiments 57 to 62, wherein the antibody is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α-beta 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.

[0291] Embodiment 64. A formulated antibody moiety composition according to any one of Embodiments 57 to 63, wherein the antibody moiety is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0292] Embodiment 65. A formulated antibody partial composition according to any one of Embodiments 57 to 64, wherein the polysorbate hydrolysis activity rate is reduced by at least about 20%.

[0293] Embodiment 66. A formulated antibody partial composition according to any one of Embodiments 57 to 65, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0294] Embodiment 67. A method for reducing the enzymatic hydrolysis activity rate of a composition obtained from a purification platform, comprising: subjecting a sample to a purification platform comprising, in this order, (a) a capture step including treatment by affinity chromatography, and (b) a purification step including treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step, or after the capture step and before the purification step, each deep filtration step comprising treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membranes, and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the enzymatic hydrolysis activity rate of the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps.

[0295] Embodiment 68. The method according to Embodiment 67, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

[0296] Embodiment 69. The method according to Embodiment 67 or 68, wherein the relative decrease in the enzymatic hydrolysis activity rate of the composition is at least about 20% compared to the purification of a sample using the same purification platform without a deep filtration step.

[0297] Embodiment 70. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, comprising: subjecting a sample to a purification platform comprising, in this order, (a) a capture step comprising treatment by affinity chromatography, and (b) a purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography, and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step and before the purification step, or after the purification step, each deep filtration step comprising treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone 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 the purification of a sample using the same purification platform without one or more deep filtration steps.

[0298] Embodiment 71. The method according to Embodiment 70, wherein one or more hydrolytic enzymes can hydrolyze the polysorbate.

[0299] Embodiment 72. The method according to Embodiment 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 the purification of a sample using the same purification platform without a deep filtration step.

[0300] Embodiment 73. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, comprising subjecting a sample to a purification platform comprising, in this order, (a) a capture step including treatment by affinity chromatography, and (b) a purification step including treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography and multimodal chromatography, wherein the purification platform further comprises one or more deep filtration steps, one or more of which are performed before the capture step, after the capture step, or after the capture step and before the purification step, each deep filtration step comprising treatment by a deep filter, the deep filter comprising (i) silica and polyacrylic fibers, (ii) hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membranes, and (iii) cellulose fibers, diatomaceous earth, and perlite, thereby reducing the degradation of polysorbate in the composition compared to the purification of a sample using the same purification platform without one or more deep filtration steps.

[0301] Embodiment 74. The method according to Embodiment 73, wherein the relative reduction in the degradation of polysorbate in the composition is at least about 5% compared to the purification of a sample using the same purification platform without a deep filtration step.

[0302] Embodiment 75. The method according to any one of Embodiments 67 to 74, wherein the deep filter containing silica and polyacrylic fibers contains a silica filter aid and polyacrylic fiber pulp.

[0303] Embodiment 76. The method according to any one of Embodiments 67 to 74, wherein the deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane comprises four layers comprising the hydrogel Q-functionalized nonwoven fabric material and a 9-zone microporous membrane.

[0304] Embodiment 77. The method according to any one of Embodiments 67 to 74, wherein the deep filter comprising cellulose fibers, diatomaceous earth, and perlite comprises two layers, each layer comprising a cellulose filter matrix, the cellulose filter matrix being impregnated with one or more filtration aids comprising diatomaceous earth or perlite, and each layer further comprising a resin binder.

[0305] Embodiment 78. The method according to any one of Embodiments 67 to 77, wherein the deep filter is selected based on the pH of the solution entering the deep filter.

[0306] Embodiment 79. The method according to Embodiment 78, wherein a deep filter containing silica and polyacrylic fibers is selected when the amount of solution entering the deep filter is about 5 to about 6.5.

[0307] Embodiment 80. The method according to Embodiment 78, wherein a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane is selected when the amount of solution entering the deep filter is about 7 to about 8.5.

[0308] Embodiment 81. The method according to any one of Embodiments 67 to 80, further comprising the step of selecting a deep filter based on the pH of the solution that enters the deep filter.

[0309] Embodiment 82. The method according to any one of Embodiments 67 to 81, wherein the purification platform comprises, in this order, a deep filtration step including treatment with a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane, a capture step including treatment with protein A chromatography, and a purification step.

[0310] Embodiment 83. The method according to Embodiment 82, wherein the purification step includes treatment by HIC.

[0311] Embodiment 84. The method according to Embodiment 83, wherein the HIC is phenyl SEPHAROSE® high-speed flow chromatography.

[0312] Embodiment 85. The method according to Embodiment 82, wherein the purification step includes treatment by cation exchange chromatography.

[0313] Embodiment 86. The method according to Embodiment 85, wherein the cation exchange chromatography is POROS® 50HS.

[0314] Embodiment 87. The method according to any one of Embodiments 67 to 86, wherein the purification platform further comprises a second deep filtration step, which includes treatment with a deep filter containing silica and polyacrylic fibers, the second deep filtration step being performed after the capture step and before the purification step.

[0315] Embodiment 88. The method according to Embodiment 82, wherein the purification step includes treatment by multimodal chromatography.

[0316] Embodiment 89. The method according to Embodiment 88, wherein the multimodal chromatography is Capto Adhere.

[0317] Embodiment 90. The method according to Embodiment 88 or 89, wherein the purification platform further comprises a second deep filtration step including treatment with a deep filter comprising a hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane, the second deep filtration step being performed after the capture step and before the purification step.

[0318] Embodiment 91. The method according to any one of Embodiments 67 to 90, wherein the purification platform is for the purification of a target from a sample, and the sample contains a target and one or more host cell impurities.

[0319] Embodiment 92. The method according to Embodiment 91, wherein the target comprises a polypeptide.

[0320] Embodiment 93. The method according to Embodiment 91 or 92, wherein the host cell impurity is a host cell protein.

[0321] Embodiment 94. The method according to any one of Embodiments 67 to 93, wherein the purification platform further includes a virus inactivation step, the virus inactivation step being performed after the capture step.

[0322] Embodiment 95. The method according to Embodiment 94, wherein one or more deep filtration steps are performed after the virus inactivation step.

[0323] Embodiment 96. The method according to any one of Embodiments 67 to 95, wherein the purification platform further includes an ultrafiltration / diafiltration (UFDF) step, the UFDF step being performed after the purification step.

[0324] Embodiment 97. The method according to any one of Embodiments 67 to 96, further comprising determining the enzymatic hydrolysis activity rate of the composition.

[0325] Embodiment 98. The method according to any one of Embodiments 67 to 97, further comprising determining the level of one or more hydrolytic enzymes in the composition.

[0326] Embodiment 99. The method according to any one of Embodiments 67 to 98, wherein the composition comprises a polysorbate.

[0327] Embodiment 100. The method according to Embodiment 99, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0328] Embodiment 101. The method according to any one of Embodiments 67 to 100, further comprising a sample processing step.

[0329] Embodiment 102. The method according to any one of Embodiments 67 to 101, wherein the sample is a cell culture sample or derived from a cell culture sample.

[0330] Embodiment 103. The method according to Embodiment 102, wherein the cell culture sample comprises host cells, and the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells.

[0331] Embodiment 104. The method according to any one of Embodiments 67 to 103, wherein the sample comprises a host cell or a component derived therefrom.

[0332] Embodiment 105. The method according to any one of Embodiments 67 to 104, wherein the sample comprises one or more host cell proteins, and one of the one or more host cell proteins is a hydrolase.

[0333] Embodiment 106. The method according to Embodiment 105, wherein the hydrolytic enzyme is lipase, esterase, thioesterase, phospholipase, or ceramidase.

[0334] Embodiment 107. The method according to any one of Embodiments 67 to 106, wherein the sample comprises a target, and the target is an antibody portion.

[0335] Embodiment 108. The method according to Embodiment 107, wherein the antibody portion is a monoclonal antibody.

[0336] Embodiment 109. The method according to Embodiment 107 or 108, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

[0337] Embodiment 110. The method according to any one of Embodiments 107 to 109, wherein the antibody portion is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-abeta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-Her2 / CD3 antibody, anti-FGFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

[0338] Embodiment 111. The method according to any one of Embodiments 107 to 110, wherein the antibody portion is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

[0339] Embodiment 112. A pharmaceutical composition obtained by the method described in any one of Embodiments 67 to 111.

[0340] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present disclosure is further illustrated by the following embodiments, but these embodiments should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described therein. [Examples]

[0341] Example 1 This example demonstrates a comparison between two purification platforms for purifying the antibody, trastuzumab, from unprepared bulk, using: (1) a general purification platform, and (2) an identical purification platform that includes an additional PDD1 deep filtration step performed after preparing the eluate from affinity chromatography and before cation exchange (CEX) chromatography.

[0342] A general purification platform (1) was implemented in two consecutive sets, consisting of the following sequential steps: affinity chromatography, eluate conditioning, cation exchange chromatography, anion exchange chromatography, and tangential flow filtration and conditioning of the pool obtained from anion exchange chromatography. The names and descriptions of the pools in the purification process were as follows in Table 1. TIFF2026071220000001.tif41170

[0343] A purification platform with an added PDD1 deep filtration step (2) was used in two consecutive sets. The hydrolysis activity of polysorbate at the unadjusted bulk level was compared by free fatty acid mass spectrometry (FAMS), and the methodology is disclosed in more detail in the Materials and Methods section.

[0344] For purification platforms including deep filtration, the eluate was conditioned after affinity chromatography and before filtering with a PDD1 deep filter (Pall PDD1; SUPRAcap(trademark)-50 SC050PDD1 (lot: 102992583); area: 22 cm2). The PDD1 deep filter was equilibrated with CEX equilibration buffer. The filtration of the conditioned affinity pool was pressure controlled. The filtration process was performed at ambient temperature (15°C to 30°C). Trastuzumab was passed through the PDD1 filter. The PDD1 deep filter was washed with CEX equilibration buffer before use and after filtration. The PDD1 deep filter was discarded after each use. The acceptable range for the CEX equilibration buffer was as follows: 0.020~0.040 M MES (2-(N-morpholino)ethanesulfonic acid), 0.042~0.048 M NaCl, pH 5.50~5.70, and conductivity 5.10~5.70 mS / cm. The operating conditions for the PDD1 deep filter were as shown in Table 2. TIFF2026071220000002.tif32170

[0345] Cation exchange chromatography (SP Sepharose® FF chromatography) was performed in binding elution mode. The cation exchange step reduces 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 a gradually increasing sodium chloride concentration gradient, and trastuzumab was eluted using stepwise elution. All chromatography steps were performed at ambient temperature (15°C to 30°C).

[0346] Before loading onto the cation exchange column, the affinity pool was conditioned by adjusting the pH to 5.5 ± 0.3 with tris(hydroxymethyl)aminomethane (Tris) base. If the pool was titrated in excess, the pool was adjusted to a specific pH with citrate, followed by the addition of highly purified water (if necessary) to adjust the conductivity to 3.5 ± 1.0 mS / cm. The cation exchange column was equilibrated with equilibration buffer and loaded with the conditioned affinity pool. After loading, the column was washed with equilibration buffer, then with a gradient wash to increase conductivity, followed by another wash with equilibration buffer. Trastuzumab was eluted from the column by stepwise elution with elution buffer. Elution collection was initiated and terminated based on absorbance and volume.

[0347] The acceptable range for the CEX equilibration buffer was as follows: 0.020–0.040 M MES, 0.042–0.048 M NaCl, pH 5.50–5.70, and conductivity 5.10–5.70 mS / cm. The acceptable range for the elution buffer was as follows: 0.020–0.040 M MES, 0.092–0.098 M NaCl, pH 5.50–5.70, and conductivity 10.10–10.80 mS / cm. The operating conditions for cation exchange chromatography were as shown in Table 3. TIFF2026071220000003.tif72170 a Grams trastuzumab / liter SP Sepharose® cation exchange resin.

[0348] Anion exchange chromatography (Q Sepharose® chromatography) was performed in flow-through mode to reduce CHO HCP, DNA, protein A, and potential viruses. Under the loading and washing conditions used, trastuzumab flowed through the column. All chromatography steps were performed at ambient temperature (15°C to 30°C).

[0349] The pH of the cation exchange pool was adjusted to 8.0 ± 0.5 using Tris base and, if necessary, MES, and the conductivity was adjusted to 5.5–7.8 mS / cm with high-purity water. The anion exchange column was equilibrated with equilibration buffer and then loaded into the pH-adjusted cation exchange pool. After loading was complete, the column was washed with equilibration buffer. Pooling was based on absorbance and volume. The pH of the anion exchange pool was adjusted to 6.0 ± 0.1 with acetic acid.

[0350] The acceptable range for the equilibration buffer was as follows: 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 were as shown in Table 4. TIFF2026071220000004.tif50170

[0351] Tangential flow filtration (TFF) of a conditioned anion exchange pool was performed for concentration and diafiltration. To achieve a protein concentration of 30 ± 5 mg / mL for the unconditioned bulk, the conditioned anion exchange pool was concentrated using a TFF device equipped with a 30 kDa polyethersulfone (PES) membrane. Subsequently, the buffer composition was adjusted to meet the requirements by adding a histidine-containing solution.

[0352] Before use, the ultrafiltration membrane was equilibrated with diafiltration buffer. The conditioned anion exchange pool was concentrated to an intermediate concentration of 10–50 g / L, and diafiltration was performed in the TFF unit using a minimum of 8 pool volumes of diafiltration buffer. Subsequently, the buffer composition was adjusted to 0.02 mol / L histidine / histidine HCl, pH 5.3 ± 0.2 by adding a corresponding amount of conditioning buffer. If necessary, the protein concentration was adjusted to 30 ± 5 mg / mL by adding diafiltration buffer.

[0353] The diafiltration 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. TIFF2026071220000005.tif30170

[0354] The amount of host cell protein in the cation exchange chromatography load composition was measured, and the results are provided in Table 6. Compared to a general purification platform, a decrease in host cell protein levels was observed for both replicas from the purification platform with the PDD1 deep filter. TIFF2026071220000006.tif41170

[0355] The amount of host cell protein in the TFF pool after conditioning by diafiltration was measured, and the results are shown in Table 7. Compared to a general purification platform, a decrease in host cell protein levels was observed in both replicas from the purification platform with the PDD1 deep filter. TIFF2026071220000007.tif58170

[0356] Hydrolytic activity in the TFF pool after conditioning by diafiltration was measured, and the results are shown in Figure 2. Hydrolytic activity was measured using FAMS at 40°C with a final trastuzumab concentration of 6 g / L, 0.04% (w / v) SR-PS20, 10 mM methionine, and 100 mM Tris pH 8.0. A decrease in the enzymatic hydrolytic activity rate, indirectly measured by the amount of free fatty acids, was observed in both replicas from the purification platform with the PDD1 deep filter compared to replicas from a general purification platform (Figure 2). Materials and methods

[0357] Determination of protein concentration. Protein concentration was determined by UV spectroscopy using either a Cary® 50 UV-Vis spectrophotometer (Varian) or a NanoDrop® OneC (Thermo Scientific). Protein samples were diluted with their respective buffers and measured in double sets. The concentration was determined according to the following formula derived from the Lambert-Beer law: c = (A280nm~A320nm) / ε·d·F, protein concentration [mg / ml], absorbance, ε extinction coefficient [ml / (mg·cm)], d cell length [cm], and F dilution coefficient. The specific extinction coefficients for trastuzumab, falisimab, and FAP-IL2v are 1.48, 1.7, and 1.35 ml / (mg·cm), respectively.

[0358] Lipase activity assay (LEAP assay). The lipase activity assay measured lipase activity by monitoring the conversion of a non-fluorescent substrate (4-MU, Chem Impex Int'l Inc) to a fluorescent product (MU, Sigma-Aldrich) by cleavage of the substrate ester bond. The protein pool sample to be analyzed was rebuffered to 150 mM Tris-Cl pH 8.0 using an Amicon Ultra-0.5 ml centrifugal filter unit (10,000 Da cutoff, Merck Millipore). The assay reaction mixture contained 80 μL of reaction buffer (150 mM Tris-Cl pH 8.0, 0.25% (w / v) Triton X-100 and 0.125% (w / v) gum arabic), 10 μL of 4-MU substrate (1 mM in DMSO), and 10 μL of protein pool sample. The protein pool sample concentration was adjusted from 10 to 30 g / L and tested at three different concentrations. Each reaction was set up in three technical iterations on a 96-well half-area polystyrene plate (black transparent flat-bottom with lid, Corning Incorporated), and the increase in fluorescence signal (excited at 355 nm, emitted at 460 nm) was monitored every 10 minutes by incubating the reaction plate at 37°C for 2 hours using an Infinite 200Pro plate reader (Tecan Life Sciences). The MU generation rate was determined from the slope over fluorescence time (0.5 hours to 2 hours), and the raw rate of the reaction (k) was determined. raw [RFU / h]) represents

[0359] The enzyme blank reaction was further set up to measure the non-enzymatic cleavage of the substrate induced by the buffer matrix. 10 μL of protein pooled sample was replaced with 10 μL of 150 mM Tris-Cl pH 8.0 in the reaction mixture. Autocleavage rate (k selfThe cleavage [RFU / h] was determined from the slope of the fluorescence over time (0.5 h to 2 h). To convert the fluorescence signal (RFU) to μM of MU, a standard MU triplicate was added per plate. 10 μL of MU (100 μM in DMSO) was supplemented with 10 μL of 150 mM Tris-Cl pH 8.0 and 80 μL of reaction buffer. The conversion factor a [RFU / μM] was calculated by averaging the fluorescence signal (0.5 h to 2 h) and dividing by the final concentration of MU present in the well.

[0360] The reaction rate (k raw [RFU / h]) of the sample was subtracted from the reaction rate (k self -cleavage [RFU / h]) of the enzyme blank, and the terms were divided by the conversion factor a [RFU / μM] to convert the fluorescence signal to μM MU / h, thereby determining the lipase activity of the sample in [μM MU / h]. The activity was normalized against the protein concentration applied per well. To report the hydrolysis activity as a percentage, the lipase activity of the reference sample was set to 100%.

[0361] 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 the PS20 stability study and subsequently analyzed by mass spectrometry. Unless otherwise specified, protein pool samples were adjusted to the same protein concentration (as shown in the description of each experiment) containing 0.04% (w / v) SR-PS20, 10 mM L-methionine, and 100 mM Tris pH 8. L-methionine was added as an efficient antioxidant to control the oxidative degradation of PS20 during the course of the experiment. As a buffer control sample, the applied protein volume was replaced with the same volume of the corresponding elution buffer system.

[0362] All reaction mixtures were incubated in a Thermomixer (Eppendorf) at either 37 °C or 40 °C, with shaking at 600 rpm. Samples were removed at defined time points (as shown in each figure) and stored at -80 °C until further analysis.

[0363] 50 μL of the sample was transferred to a new Eppendorf cup. 200 μL of FFA dissolution solution (500 ng / mL of D in acetonitrile) was added. 23 Lauric acid and 500 ng / mL 13 C 14 Myristic acid was added and vortexed for a short time. The sample was centrifuged at 14,000 rpm for 5 minutes and transferred to an HPLC vial for MS analysis. Separation of fatty acids from 5 μL of injection sample was performed on a Thermo Scientific® Vanquish® UHPLC system using an ACQUITY UPLC® Peptide BEH C18 column (1.7 μm 2.1 × 150 mm and 300 A). Eluent A (0.1% aqueous ammonium hydroxide solution) and eluent B (100% acetonitrile) were used with a flow rate of 0.3 mL / min and a column temperature of 60°C in the following gradient. The initial condition was 70% eluent B. The gradient was changed linearly from 0.2 min to 5.5 min, increasing eluent A to 100% and holding up to 6.0 min. Eluent B was set to 70% at 6.1 min and held up to 10.0 min for equilibration. A mass spectrometer (Triple TOF® 6600, AB Sciex) was operated in negative ionization mode with an ion spray voltage of -4500V. The source temperature was set to 450°C, and the TOF mass range was set to 100-1000 m / Z. The decay potential was -120V, and the collision energy was -10V.

[0364] Lauric acid, myristic acid, and isotope-labeled (D 23 )-Lauric acid and ( 13 C 14 XIC was generated with respect to the mass of myristic acid. Each peak was integrated to obtain the values ​​of lauric acid and D. 23 - The peak area ratio between lauric acid and myristic acid and 13 C 14 - The ratio between lauric acid and myristic acid was determined. The concentrations of lauric acid and myristic acid in the sample were calculated using the peak area ratio. The measurement was performed in two series. The amount of the reference sample was set to 100% in order to report the amount of FFA (lauric acid (LA) and myristic acid (MA)) as a percentage. Example 2

[0365] This example demonstrates a comparison between three platforms for purifying the antibody trastuzumab, using: (1) a general purification platform; (2) a general purification platform with the addition of a PDD1 deep filtration step performed after preparing the eluate from affinity chromatography and before cation exchange (CEX) chromatography; and (3) a general purification platform with the addition of an EMPHAZE® deep filtration step performed after preparing the eluate from affinity chromatography and before cation exchange (CEX) chromatography.

[0366] A typical purification platform (1) consisted of: affinity chromatography, eluate conditioning, cation exchange chromatography, anion exchange chromatography, and tangential flow filtration, as well as conditioning of the pool obtained from anion exchange chromatography.

[0367] For the purification platform including the deep filtration step, the eluate was conditioned after affinity chromatography and before filtering through the deep filter. The PDD1 deep filter used was Pall PDD1, SUPRAcap(trademark)-50 SC050PDD1 (lot 102992583), area: 22 cm². The EMPHAZE(trademark) deep filter used was EMPHAZE(trademark) AEX Hybrid (lot: S210650302), area: 25 cm². Trastuzumab flows through the deep filter. The filtration step is performed at ambient temperature (15°C to 30°C).

[0368] Before use, the deep filter was equilibrated with cation exchange equilibrium buffer. Filtration of the conditioned affinity pool was flow-controlled. No rinsing was performed after filtration. This allowed for the examination of the actual reduction of host cell proteins (enzymes). The filter was discarded after each use. The acceptable range for the CEX equilibrium buffer was as follows: 0.020–0.040 M MES, 0.042–0.048 M NaCl, pH 5.50–5.70, and conductivity 5.10–5.70 mS / cm. The operating conditions for the deep filter were as shown in Table 8. TIFF2026071220000008.tif55170

[0369] The amount of host cell protein in the cation exchange chromatography load composition was measured, and the results are provided in Table 9. Protein concentration was measured according to Example 1. Compared to a general purification platform, a decrease in host cell protein levels was observed in both the purification platform equipped with the EMPHAZE® deep filter and the purification platform equipped with the PDD1 deep filter. TIFF2026071220000009.tif32170

[0370] The lipase activity of a cation exchange chromatography load on a typical platform (after affinity chromatography, without deep filtration, see reference) was compared to a corresponding cation exchange chromatography load that included an additional deep filtration step after affinity chromatography (Figure 3). The lipase activity assay was performed according to Example 1.

[0371] The hydrolytic activity of a cation exchange chromatography load on a typical platform (after affinity chromatography, without deep filtration, see reference) was compared to a corresponding cation exchange chromatography load including an additional deep filtration step after affinity chromatography (Figure 4). FAMS analysis was performed according to Example 1. Hydrolytic activity was measured using FAMS at 40°C, 0.04% (w / v) SR-PS20, 10 mM methionine, 100 mM Tris pH 8.0, and a final trastuzumab concentration of 4.8 g / L. A decrease in the rate of enzyme hydrolysis activity, as measured by lipase activity assay (Figure 3), and a decrease in the amount of FAA in the FAMS assay (Figure 4) were observed for both purification platforms including deep filtration compared to the typical purification platform. Example 3

[0372] This example demonstrates the use of a purification platform for purifying anti-VEGF / Ang2 antibodies using a deep filtration step performed on HCCF (recovered cell fluid) before sterilization, and a second deep filtration step performed after affinity chromatography (CaptureSelect® FcXL). The purification platform used is detailed in Figure 5. A reference control was performed using the illustrated purification process without the additional deep filtration step of HCCF (Figure 5).

[0373] As shown in Figure 5, HCCF was filtered through three different deep filters designed to remove potential host cell proteins. Antibodies flowed through the filters. The filtration process was carried out at ambient temperature (15°C to 30°C).

[0374] Before use, the three deep filters were equilibrated using affinity equilibrium buffer for EMPHAZE® (EMPHAZE® AEX Hybrid (Lot: S228585702), Area: 25cm²), VR02 (BioCap_VR02 (Lot: 3923452), Area: 25cm²), and 120ZB (BioCap_120ZB (Lot: 3923452), Area: 25cm²), and cation exchange equilibrium buffer for PDD1 (SUPRAcap®-50 SC050PDD1 (Lot: 103119429), Area: 22cm²). Filtration of HCCF was pressure controlled (feed pressure = 0.2MPa; maximum supply flow: 25mL / min). Filtration of the conditioned affinity pool was flow controlled (supply flow = 5.2ml / min; pressure control: 0.2MPa). After filtration, the filter was washed with the same buffer solution to recover the product. The filter was discarded after each use. The equilibration buffer solutions are provided in Table 10. TIFF2026071220000010.tif35170

[0375] PDD1 filter flush, 200 mL (approximately 90 L / m³) 2 The procedure was performed using sterile water for injection. EMPHAZE® filter flush was administered using 200 mL (approximately 90 L / m³). 2 The procedure was performed using C1 equilibration buffer. The volume of filtered HCCF was 1750 mL (approximately 700 L / m2 for EMPHAZE®).

[0376] The operating conditions for CaptureSelect FcXL are as shown in Table 11. TIFF2026071220000011.tif52170

[0377] Host cell protein loading was measured at various time points on the purification platform (see Figure 5), and the results are shown in Table 12. TIFF2026071220000012.tif71170

[0378] Hydrolytic activity was measured by the lipase activity assay described in Example 1. The decrease in the enzymatic hydrolytic activity rate of FcXL eluate from various purification platforms was compared to any of the deep filters tested (EMPHAZE) compared to a general purification platform without additional deep filtering (Figure 6A). (商標) A decrease in the enzymatic hydrolysis activity rate of the PDD1 filtrate of various purification platforms was observed with any of the deep filters tested (EMPHAZE) compared to a general purification platform without additional deep filters (Figure 6B). (商標) Observed in VR02, 120ZB.

[0379] FAMS assays were performed to compare two purification platforms for antibody purification with a strong cation exchange chromatography pool: (1) a standard purification platform, and (2) an identical purification platform including an additional 120 ZB deep filtration step performed before affinity chromatography. The standard purification platform consisted of the following sequence of steps: affinity chromatography, eluate conditioning, deep filtration, multimodal anion exchange chromatography, strong cation exchange chromatography, and tangential flow filtration. The FAMS assays were performed according to Example 1 and 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. A decrease in the rate of enzyme hydrolysis activity, measured by the amount of free fatty acids, was observed in the purification platform with the 120 ZB deep filter compared to the standard purification platform (Figure 7). Example 4

[0380] This example demonstrates a comparison of purification platforms for purifying anti-FAP-IL2v, incorporating two different deep filters (X0SP or PDD1) for filtering conditioned affinity chromatography (protein A chromatography) eluates.

[0381] As described in Example 1, an HCCF sample was prepared and filtered.

[0382] The lipase activity of protein A chromatography eluates from a general platform (without deep filtration) was compared to that of affinity chromatography eluates obtained by deep filtration using a purification platform that includes either an X0SP or PDD1 deep filtration step performed on the protein A chromatography eluate. The lipase activity assay was performed according to Example 1. The results of the lipase activity of the fraction obtained from the X0SP deep filter are shown in Figure 8A. The results of the lipase activity obtained from the PDD1 deep filter are shown in Figure 8B. Example 5

[0383] This example demonstrates purification optimization experiments for the purification of various antibody moieties, conducted to find options that minimize the hydrolysis of polysorbate in antibody moieties obtained from a purification platform. The experiments disclosed herein evaluate the inclusion of deep filters such as EMPHAZE® as load filters for protein A and a second chromatography column, as well as HIC media (SARTOBIND® phenyl membrane) as a polished column elution pool filter or load filter for a subsequent viral filtration step.

[0384] The potential of several deep filters, including EMPHAZE® and X0SP, to remove or reduce hydrolytic enzymes involved in polysorbate degradation was evaluated. The incorporation of EMPHAZE® filters was evaluated at two processing levels in a typical mAb purification process flow. The first option is a protein A load filter before protein A chromatography, filtering HCCF before loading it onto the protein A column. As summarized in Table 13, relative hydrolytic activity is reduced by more than 40% when HCCF is filtered before protein A chromatography compared to the typical purification process. TIFF2026071220000013.tif48170

[0385] To demonstrate that the reduction in polysorbate degradation achieved with EMPHAZE® was not associated with a decrease in host cell protein alone, HCCF samples were purified with Protein A at increasing EMPHAZE® processing rates, and the pools were analyzed for both CHOP and polysorbate degradation activity. As shown in Figure 9, the decrease in CHOP values ​​was dependent on the EMPHAZE® filtration rate, while the significant decrease in polysorbate degradation rate was relatively constant. The significant decrease in polysorbate degradation activity achieved compared to the control was at approximately the same level as the control, despite a continuous increase in CHOP values ​​with increasing EMPHAZE® clarification rates, at 800 L / m³. 2 The processing volume remains relatively constant.

[0386] The second option evaluated was the placement of EMPHAZE® and X0SP deep filters downstream of the virus inactivation step, or as load filters for a second column chromatography step. For this evaluation, a pool of protein A from several molecules was neutralized to either pH 5.5 or pH 8.0 and filtered at 300 L / m³ with EMPHAZE® or X0SP. 2 The samples were filtered at the specified rate. The polysorbate hydrolysis activity of the filtered pool was compared to that of an unfiltered control sample. As summarized in Table 14, both the EMPHAZE® and X0SP filters showed a significant reduction in polysorbate degradation compared to the unfiltered control pool. As shown in Figure 10, the polysorbate hydrolysis activity (measured using a lipase activity assay in the purification of anti-tau mAbs in the protein A pool) was dependent on the filtering rate of the X0SP deep filter at pH 5.5. TIFF2026071220000014.tif36170

[0387] As an approach to reduce polysorbate degradation, the use of HIC membranes was evaluated. HIC membranes can be placed after the final polypeptide chromatography column step (e.g., anion exchange chromatography), after a pH holding step (e.g., pH 5-6) performed before viral filtration, and / or after a viral filtration step performed before a UFDF step.

[0388] The primary HIC membrane evaluated in this study was a SARTOBIND® phenyl membrane. The final polypeptide chromatography pool, after being adjusted to various pH values, was filtered through a SARTOBIND® membrane filter. The filtered pool was analyzed for relative polysorbate degradation. As shown in Figures 11A-11C, relative polysorbate activity was significantly reduced compared to the control. The same data also show that the decrease in activity depends on the membrane volume throughput (Figures 11A-11C). Specifically, Figure 11A shows the specific activity of polysorbate degradation for ocrelizumab at different throughputs at pH 5.5. Figure 11B shows the specific activity of polysorbate degradation for sericrelumab at different throughputs at pH 5.5. Figure 11B shows the specific activity of polysorbate degradation for tocilizumab at different throughputs at pH 6.5 (Figure 11C). Materials and methods

[0389] Resins Pro A Sepharose® FF, Fractogel® TMAE, and ceramic hydroxyapatite resin were purchased from GE Healthcare (Uppsala, Sweden), Tosoh Biosciences (King of Prussia, Pennsylvania), and Bio-Rad (Hercules, California), respectively. Amicon centrifugal filters and X0SP deep filters were obtained from Millipore (Bedford, Massachusetts). EMPHAZE® AEX deep filters were obtained from 3M (Meriden, Connecticut), and SARTOBIND® phenyl membranes were obtained from Sartorius (Bohemia, New York). 4-Methylumbelliferyl caprilate, Triton® X-100, and gum arabic were obtained from Research Organcis (Cleveland, Ohio) and Acros Organics (Bridgewater, New Jersey), respectively. The ultra-purified (SR) grade PS20 used in the lipase activity assay was manufactured by Croda (Newark, New Jersey). All monoclonal antibodies reported here were humanized or human IgG1 expressed in CHO cells and produced in Roche (South San Francisco or Oceanside, California).

[0390] For small-scale EMPHAZE (trademark) and other deep filters, 25cm 2 A capsule of size was used. The filter was first washed with 25 mM Tris, 250 mM NaCl, pH 7.5 at a flow rate of 100 L / m² and 8 ml / min. After equilibration, either HCCF (Pro A loaded) or a neutralized protein A elution pool was added at a rate of 800 L / m². 2 It was filtered up to this point. The fraction was divided into 100 L / m³. 2 Each sample was collected, purified through subsequent column processes, and its polysorbate degradation activity was measured.

[0391] A 3 ml device was used to evaluate the SARTOBIND® phenyl membrane. The membrane was first washed with 30 ml of equilibration buffer at a flow rate of 15 L / min. After equilibration, the final chromatography pool was filtered with the SARTOBIND® phenyl membrane, the fraction was collected, and the relative polysorbate degradation activity was assayed.

[0392] All small-scale chromatography was performed on a 0.66 cm × 20 cm column. For protein A purification, the column was first pre-equilibrated in equilibration buffer, and HCCF was loaded onto a resin at a concentration of 10-20 g / L. After loading, the column was washed with more than 3 column volumes of equilibration buffer and more than 4 column volumes of washing buffer. The bound protein was eluted with 2.5 mM HCl pH 2.7 or 150 mM acetic acid. The eluted fractions with a pH of 0.5 OD to 0.5 OD were collected, neutralized to pH 5, and assayed for polysorbate degradation activity.

[0393] Lipase enzyme activity was investigated by tracking the cleavage of ester bonds in umbelliferyl substrates with a structure similar to polysorbate. In this study, a reaction mixture was prepared by mixing 10 μL of 10 mM 4-methylumbelliferyl caprylate in DMSO with 80 μL of reaction buffer (50 mM Tris, pH 8.0, 0.4% Triton® X-100 and 0.1% gum arabic). Fluorescence excitation and emission wavelengths were set to 355 nm and 460 nm, respectively. Fluorescence dynamics were continuously monitored at 37°C for 2–4 hours. Lipase activity for each sample was determined by calculating the initial reaction rate for kinetic hardening using linear fitting, and the reaction rate in buffer only was corrected to account for background hydrolysis. Specific activity was determined by dividing the reaction rate by the sample protein concentration.

[0394] Protein samples were sterile filtered through a 0.2 μm fluorozine syringe filter and spiked with 25x conditioning buffer (20 mg / mL methionine in 10 mM histidine acetate pH 5, 1% w / v SR PS20). The added samples were dispensed into Eppendorf tubes under sterile conditions and incubated at 25°C for up to 20 days. Aliquots were taken at each time point and frozen at -70°C until free fatty acid extraction was performed.

[0395] Free fatty acids in each sample were extracted using an acetonitrile solution containing an isotope-labeled FFA internal standard. After centrifugation at 14000 rpm for 5 minutes, the supernatant was transferred to an HPLC vial with a glass insert and frozen until measurement. A Waters H-class Bio UPLC system equipped with a Waters ACQUITY UPLC® BEH300 C18 (1.7 μm, 2.1 × 150 mm) column was used in combination with an AB Sciex 6600 mass spectrometer for FFA detection. Separation of free fatty acids from 5 μL of injected sample was performed using 5 mM ammonium acetate and 0.1% ammonium hydroxide in water as buffer A and 100% acetonitrile as buffer B, at a flow rate of 0.3 mL / min and a column temperature of 60°C. This method was started with 70% buffer B for 0.2 minutes, followed by a gradient to 100% B over 5.3 minutes, then back to 70% B over 0.1 minutes, and equilibrated at 70% B for 3.9 minutes. The mass spectrometer was operated in negative ionization mode with an ion spray voltage of -4500V. The source temperature was set to 450°C, and the top mass range was set to 100-1000 m / z. The declustering potential was -120V, and the collision energy was -10V. The accumulation of FFA in the linear range was fitted to a linear regression to calculate the initial hydrolysis rate.

[0396] Host cell proteins in all in-process samples were measured using the in-house CHO protein (CHOP) ELISA assay, and DNA was quantified using the in-house qPCR method. Example 6

[0397] This example demonstrates a comparison between the following two purification platforms for antibody purification, the purification platforms comprising: (1) deep filtration of HCCF using a 120ZB10A deep filter, followed by protein A chromatography, or (2) deep filtration of HCCF using an EMPHAZE® AEX deep filter, followed by protein A chromatography.

[0398] Cell culture media (HCCF) were collected individually from three different cell cultures for the expression of three different antibody moieties (AM1, AM2, and AM3). For a purification platform (Purification Platform (1)) with a 120ZB10A deep filtration step, each HCCF pool was subjected to 120ZB10A deep filtration (300 L / m³). 2 The samples were subjected to ) separately, and then to protein A chromatography. Aliquots were collected from the pool after protein A chromatography. The polysorbate hydrolysis activity of the aliquots was measured using FAMS (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The measured specific FAMS rates of the aliquots after protein A chromatography are shown in Figure 12. Aliquots from the HCCF pool (before deep filtration) were used to obtain control measurements.

[0399] For the purification platform (purification platform (2)) having an EMPHAZE® AEX deep filtration process, each HCCF pool is subjected to EMPHAZE® AEX deep filtration (300 L / m³). 2 The samples were subjected to ) separately, and then to protein A chromatography. Aliquots were collected from the pool after protein A chromatography. The polysorbate hydrolysis activity of the aliquots was measured using FAMS (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The measured specific FAMS rates of the aliquots after protein A chromatography are shown in Figure 12. Aliquots from the HCCF pool (before deep filtration) were used to obtain control measurements. Example 7

[0400] This example demonstrates a comparison between the following two purification platforms for antibody purification, the purification platforms comprising: (1) Protein A chromatography of HCCF followed by activated carbon (40CR) filtration followed by cation exchange chromatography using POROS® 50HS, or (2) Protein A chromatography of HCCF followed by deep filtration using X0SP deep filter followed by HIC using phenyl SEPHAROSE® high-speed flow.

[0401] Cell culture media (HCCF) were collected individually from three different cell cultures for the expression of three different antibody moieties (AM1, AM2, and AM4). For the purification platform without a deep filtration step (purification platform (1)), the three HCCF samples were subjected to protein A chromatography separately. The protein A chromatography pools were conditioned by adjusting the pH to 5.5 ± 0.3 using tris(hydroxymethyl)aminomethane (Tris) base, and then the conditioned pools were filtered separately using 40CR filtration (300 L / m³). 2 The samples were subjected to 40CR filtration, followed by POROS® 50 HS chromatography. Aliquots were collected from the pool after 40CR filtration and from the pool after POROS® 50 HS filtration. The polysorbate hydrolysis activity of the aliquots was measured using FAMS (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The specific FAMS rates measured for the aliquots from the pool after 40CR filtration and from the pool after POROS® 50 HS chromatography are shown in Figures 13 and 14, respectively. A control measurement was obtained using an aliquot from the Protein A chromatography pool (without 40CR filtration).

[0402] For a purification platform (Purification Platform (2)) with an X0SP deep filtration step, three HCCF samples were subjected to protein A chromatography separately. The protein A chromatography pools were conditioned by adjusting the pH to 5.5 ± 0.3 using tris(hydroxymethyl)aminomethane (Tris) base, and then the conditioned pools were separately filtered using X0SP (300 L / m³). 2 The samples were subjected to X0SP filtration, followed by phenylSEPHAROSE® high-speed flow chromatography. Aliquots from the pool after X0SP filtration and phenylSEPHAROSE® high-speed flow chromatography were collected. The polysorbate hydrolysis activity of the aliquots was measured using FAMS (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The specific FAMS rates measured for the aliquots from the pool after X0SP filtration and phenylSEPHAROSE® high-speed flow chromatography are shown in Figures 13 and 14, respectively. A control measurement was obtained using an aliquot from the Protein A chromatography pool (without X0SP deep filtration). Example 8

[0403] This example demonstrates a comparison between the following three purification platforms for antibody purification, the purification platforms being: (1) Protein A chromatography of HCCF followed by activated carbon (40CR) filtration followed by multimodal chromatography using a Capto Adhere; (2) Protein A chromatography of HCCF followed by deep filtration using an EMPHAZE® deep filter followed by multimodal chromatography using a Capto Adhere; or (3) Protein A chromatography of HCCF followed by deep filtration using a PDD1 deep filter followed by multimodal chromatography using a Capto Adhere.

[0404] Cell culture media (HCCF) were collected individually from three different cell cultures for the expression of three different antibody moieties (AM1, AM2, and AM4). For the purification platform without a deep filtration step (purification platform (1)), the three HCCF samples were subjected to protein A chromatography separately. The protein A chromatography pool was conditioned by adjusting the pH to 8.0 ± 0.5 using Tris bases, and then the conditioned pool was subjected to 40CR deep filtration (300 L / m³). 2 The samples were then subjected to CaptoAdhere chromatography separately. Aliquots were collected from the pool after 40CR filtration. The polysorbate hydrolysis activity of the aliquots was measured using a LEAP assay (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The specific LEAP rates measured for aliquots from the pool after 40CR filtration are shown in Figure 15. Aliquots from the Protein A chromatography pool (without 40CR filtration) were used to obtain control measurements.

[0405] Three HCCF samples were subjected to protein A chromatography separately using a purification platform (purification platform (2)) with an EMPHAZE® deep filtration process. The protein A chromatography pool was conditioned by adjusting the pH to 8.0 ± 0.5 using Tris bases, and then the conditioned pool was subjected to EMPHAZE® deep filtration (300 L / m³). 2 The samples were then subjected to CaptoAdhere chromatography separately. Aliquots were collected from the pool after deep filtration with EMPHAZE®. The polysorbate hydrolysis activity of the aliquots was measured using a LEAP assay (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). Figure 15 shows the specific LEAP rates measured for aliquots from the pool after deep filtration with EMPHAZE®. Aliquots from the Protein A chromatography pool (without deep filtration with EMPHAZE®) were used to obtain control measurements.

[0406] For a purification platform (Purification Platform (3)) with a PDD1 deep filtration step, three HCCF samples were subjected to protein A chromatography separately. The protein A chromatography pool was conditioned by adjusting the pH to 8.0 ± 0.5 using Tris base, and then the conditioned pool was subjected to PDD1 deep filtration (300 L / m³). 2 The samples were then subjected to Capto-Adhere chromatography separately. Aliquots were collected from the pool after PDD1 deep filtration. The polysorbate hydrolysis activity of the aliquots was measured using a LEAP assay (the methodology is disclosed in more detail in the Materials and Methods section of Example 1). The specific LEAP rates measured for aliquots from the pool after PDD1 deep filtration are shown in Figure 15. Aliquots from the Protein A chromatography pool (without PDD1 deep filtration) were used to obtain control measurements. Example 9

[0407] This example demonstrates the evaluation and comparison of workflows for purifying TYRP1 TCB antibody (for example, disclosed in its entirety in PCT / EP2019 / 08614, which is incorporated herein by reference), the workflows using different C1 deep filters followed by a C2 filtration step using a MerckMillipore Millistak+® HC Pro X0SP filter. The pre-C1 deep filters compared are deep filters containing chemically defined synthetic materials (3M® EMPHAZE® AEX Hybrid Purifier) ​​and recovery and purification deep filters (ZETA PLUS® EXT ZB series, 120ZB). Both the 120ZB and EMPHAZE® deep filters are positively charged, while the X0SP deep filter is negatively charged at pH > 4.5.

[0408] The experimental workflow and sample naming assignments are shown in Figure 16. Briefly, a cell-free sample containing TYRP1 TCB antibody with a turbidity of approximately 80 NTU was recovered from the bioreactor to be used as the loading material for the described experiment. Prior to protein A chromatography, a portion of the cell-free sample was filtered using a 120 ZB deep filter, and a second portion was filtered using an EMPHAZE® deep filter. Both filtrates were then sterile filtered and subjected to protein A chromatography using GE Healthcare's MABSELECT SURE® medium. The eluates from the 120 ZB deep filter workflow and the EMPHAZE® deep filter workflow were titrated to pH 9.0 with 1 M TRIS / HCl, pH 5.5. For each workflow, the titrated eluate is divided into aliquots. One aliquot is sterile filtered using a 0.2 μm sterile filter and kept as a reference. The second aliquot (approximately 100 mL to 120 mL) is filtered using a small Merck Millipore Millistak+(registered trademark) HC Pro X0SP filter (5 cm). 2 The samples were filtered using a filter area. Three fractions of eluate were collected from the X0SP filtration using the 120ZB workflow. Four fractions of eluate were collected from the X0SP filtration using the EMPHAZE® workflow. Each X0SP eluate fraction was then filtered using a 0.2 μm sterile filter.

[0409] Next, each aliquot obtained was analyzed by a LEAP assay according to the method provided in Example 1. A comparison of the LEAP assay results shows that both pre-C1 deep filter workflows worked well to reduce hydrolytic activity, and that the X0SP filter significantly reduced the hydrolytic activity of the capture column output (Figure 17). A comparison of the 120ZB and EMPHAZE® workflows shows that the protein A eluate from the EMPHAZE® filter material had 33% lower hydrolytic activity compared to the protein A eluate from the 120ZB filter (Figure 17). Example 10

[0410] This example demonstrates the evaluation and comparison of the purification of various HCCF samples containing TYRP1 TCB antibody (for example, disclosed in its entirety in PCT / EP2019 / 08614, which is incorporated herein by reference) using a purification platform that includes an X0SP deep filtration step performed on the protein A chromatography eluate.

[0411] 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® medium. Elutions were collected (CF 238 MSS eluate and CF 239 MSS eluate), titrated to pH 9.0 with 1 M TRIS / HCl, pH 5.5, and then subjected to X0SP deep filtration. Specifically, 35 L of CF 238 MSS eluate or 20 L of CF239 MSS eluate were filtration at 160 L / m³. 2 X0SP filter at a flow rate of / h (each 1 m 2 or 0.55 m 2 The samples were filtered using ) . In parallel, aliquots of CF 238 and CF 239 HCCF samples were purified using a reference purification platform that did not include the X0SP deep filtration step after protein A chromatography.

[0412] The lipase activity of each obtained eluate was measured as described in Example 1. The results of the lipase activity obtained from the reference and X0SP deep filters, including both the CF 238 and CF 239 purification platforms, are shown in Figures 18A (CF 238) and 18B (CF 239). As shown in Figures 18A and 18B, the hydrolytic activity of the eluate from the X0SP-containing purification platform was significantly reduced. Example 11

[0413] This example demonstrates a comparison of the purification of prasinezumab using four purification platforms incorporating various deep filtration steps for filtering conditioned affinity chromatography (protein A chromatography) eluates. Specifically, the four different deep filtration steps were based on: (i) PDD1; (ii) X0SP; (iii) PDD1 followed by X0SP; and (iv) X0SP followed by PDD1.

[0414] The affinity chromatography eluate was adjusted to pH 6.0+ / -0.2 using 2M Tris. The PDD1 and XS0P filters were equilibrated with at least 220 ml of the corresponding buffer (25 mM Tris / acetate). Both filters were loaded at less than 200 L / m2. The flow rate of the PDD1 and XS0P filters was 11 ml / min. The pressure was controlled throughout the experiment. The eluates from the deep filters were fractionated after 100 L / m2 and 200 L / m2.

[0415] The lipase activity and HCP levels in the protein A chromatography eluate from the reference approach (without deep filtration; loaded) were compared with eluates collected after each deep filtration step of the four purification platforms. Lipase activity assays were performed according to Example 1. The results of the lipase activity of the fraction obtained from the deep filter are shown in Figure 19A. The results of the HCP measurements are shown in Figure 19B.

Claims

1. A method for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, wherein the sample is (a) Capture process, and (b) Provided to the purification platform, which includes a deep filtration step, A method for thereby reducing the enzymatic hydrolysis activity rate of the composition compared to the purification of the sample using the same purification platform that does not include the deep filtration step.

2. The method according to claim 1, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

3. The method according to claim 1 or 2, wherein the relative decrease in the enzyme hydrolysis activity rate of the composition is at least about 20% compared to the purification of the sample using the same purification platform without the deep filtration step.

4. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, wherein the sample is (a) Capture process, and (b) Provided to the purification platform, which includes a deep filtration step, A method for thereby reducing the level of the hydrolytic enzyme in the composition compared to the purification of the sample using the same purification platform that does not include the deep filtration step.

5. The method according to claim 4, wherein the one or more hydrolytic enzymes can hydrolyze the polysorbate.

6. The method according to claim 4 or 5, wherein the relative decrease in the level of the one or more hydrolytic enzymes in the composition is at least about 20% compared to the purification of the sample using the same purification platform without the deep filtration step.

7. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, wherein the sample is (a) Capture process, and (b) Provided to the purification platform, which includes a deep filtration step, A method that thereby reduces the degradation of the polysorbate in the composition compared to the purification of the sample using the same purification platform that does not include the deep filtration step.

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

9. The method according to any one of claims 1 to 8, wherein the purification platform is for purifying a target from the sample, and the sample comprises the target and one or more host cell impurities.

10. The method according to claim 9, wherein the target comprises a polypeptide.

11. The method according to claim 9 or 10, wherein the host cell impurity is a host cell protein.

12. The method according to any one of claims 1 to 11, wherein the deep filtration step is performed before the capture step, or the deep filtration step is performed after the capture step.

13. The method according to any one of claims 1 to 12, wherein the deep filtration step includes processing with a deep filter.

14. The method according to claim 13, wherein the deep filter comprises a substrate containing one or more diatomaceous earth compositions, silica compositions, cellulose fibers, polymer fibers, agglomerating resins, and ash compositions.

15. The method according to claim 14, wherein at least a portion of the substrate of the deep filter includes surface modification.

16. The method according to claim 15, wherein the surface modification is one or more of quaternary amine surface modification, cationic surface modification, and anionic surface modification.

17. The method according to any one of claims 14 to 16, wherein the deep filter is selected from the group consisting of EMPHAZE® deep filter, PDD1 deep filter, ZETA PLUS® 120ZA deep filter, and ZETA PLUS® 120ZB deep filter.

18. The method according to any one of claims 1 to 17, wherein the capture step includes treatment by affinity chromatography.

19. The method according to claim 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.

20. The method according to any one of claims 1 to 19, wherein the purification platform further comprises a virus inactivation step, the virus inactivation step being performed after the capture step.

21. The method according to claim 20, wherein the deep filtration step is performed after the virus inactivation step.

22. The method according to any one of claims 1 to 21, wherein the purification platform further comprises another deep filtration step performed prior to the capture step.

23. The method according to any one of claims 1 to 22, wherein the purification platform further comprises one or more purification steps, the one or more purification steps being performed after the capture step, the deep filtration step, and, if present, the virus inactivation step.

24. The method according to claim 23, wherein one or more of the purification steps include a polypeptide purification step.

25. The method according to claim 23 or 24, wherein the purification platform further comprises another deep filtration step performed before, during, or after one or more purification steps.

26. The method according to any one of claims 1 to 25, wherein the purification platform further comprises an ultrafiltration / diafiltration (UFDF) step, the UFDF step being performed after the one or more purification steps.

27. The method according to claim 26, wherein the purification platform further comprises another deep filtration step performed before or after the UFDF step.

28. The method according to any one of claims 1 to 27, wherein the purification platform further comprises a hydrophobic interaction chromatography (HIC) purification step.

29. The method according to claim 28, wherein, if the HIC purification step is present, it is performed before, during, or after one or more of the purification steps.

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

31. The method according to claim 26 or 27, wherein the purification platform further includes a pH holding step, which is performed after the one or more purification steps, if present, and before the UFDF step.

32. The method according to claim 31, wherein the purification platform further comprises a virus filtration step, the virus filtration step being performed after the pH holding step and before the UFDF step.

33. The method according to claim 32, wherein the virus filtration step includes treatment with a virus filter.

34. The method according to claim 28, wherein the HIC purification step includes treatment with an HIC filter.

35. The method according to any one of claims 23 to 34, wherein one or more of the purification steps independently include treatment 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.

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

37. A method for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, wherein the sample is (a) Capture step including treatment by affinity chromatography, (b) Virus inactivation process, (c) Second polypeptide purification step, (d) A third polypeptide purification step, and (e) The process includes subjecting the material to a purification platform that includes an ultrafiltration / diafiltration (UFDF) step in this order. The aforementioned purification platform is as follows: (i) Before the capture step, (ii) After the capture step and before the virus inactivation step, (iii) After the virus inactivation step and before the second polypeptide purification step, (iv) After the second polypeptide purification step and before the third polypeptide purification step, (v) further comprising one or more deep filtration steps performed after the third polypeptide purification step and before the ultrafiltration / diafiltration (UFDF) step, A method for thereby reducing the enzymatic hydrolysis activity rate of the composition compared to the purification of the sample using the same purification platform that does not include the deep filtration step.

38. The method according to claim 37, wherein the purification platform further comprises, in this order, a pH holding step and a virus filtration step, performed after the third polypeptide purification step and before the UFDF step.

39. The method according to claim 38, wherein the virus filtration step includes treatment with a virus filter.

40. The aforementioned purification platform includes one or more of the following: (i) After the third polypeptide purification step and before the pH holding step, (ii) After the pH maintenance step and before the virus filtration step, (iii) After the virus filtration step and before the UFDF step, The method according to any one of claims 37 to 39, further comprising a hydrophobic interaction chromatography (HIC) purification step performed therein.

41. The method according to any one of claims 1 to 40, further comprising determining the enzyme hydrolysis activity rate of the composition.

42. The method according to any one of claims 1 to 41, further comprising determining the level of one or more hydrolytic enzymes in the composition.

43. The method according to any one of claims 1 to 42, wherein the composition comprises a polysorbate.

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

45. The method according to any one of claims 1 to 44, further comprising a sample processing step.

46. The method according to any one of claims 1 to 45, wherein the sample is a cell culture sample or derived from a cell culture sample.

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

48. The method according to any one of claims 1 to 47, wherein the sample comprises a host cell or a component derived therefrom.

49. The method according to any one of claims 1 to 48, wherein the sample comprises one or more host cell proteins, and one of the one or more host cell proteins is a hydrolase.

50. The method according to claim 49, wherein the hydrolytic enzyme is lipase, esterase, thioesterase, phospholipase, or ceramidase.

51. The method according to any one of claims 1 to 50, wherein the sample comprises a target, and the target is an antibody portion.

52. The method according to claim 51, wherein the antibody portion is a monoclonal antibody.

53. The method according to claim 51 or 52, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

54. The method according to any one of claims 51 to 53, wherein the antibody portion is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α beta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-HER2 / CD3 antibody, anti-FFFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

55. The method according to any one of claims 51 to 54, wherein the antibody portion is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

56. A pharmaceutical composition obtained by the method described in any one of claims 1 to 55.

57. A formulated antibody partial composition comprising an antibody portion and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate and a shelf life exceeding 24 months.

58. A formulated antibody partial composition comprising an antibody portion and a polysorbate, wherein the composition has a reduced polysorbate hydrolysis activity rate, the shelf life of the composition is extended compared to the shelf life indicated in documentation submitted to the health authorities relating to the formulated antibody partial composition, and the shelf life is extended by at least six months compared to the shelf life indicated in the documentation.

59. A formulated antibody partial composition comprising an antibody portion, wherein the formulated antibody partial composition exhibits reduced polysorbate degradation, the degradation being at least about 20% lower than the degradation described in documents submitted to the health authorities relating to the formulated antibody partial composition.

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

61. The formulated antibody partial composition according to any one of claims 57 to 60, wherein the antibody portion is a monoclonal antibody.

62. The formulated antibody partial composition according to any one of claims 57 to 61, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

63. The formulated antibody partial composition according to any one of claims 57 to 62, wherein the antibody is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α beta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-HER2 / CD3 antibody, anti-FFFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

64. A formulated antibody portion composition according to any one of claims 57 to 63, wherein the antibody portion is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

65. The formulated antibody partial composition according to any one of claims 57 to 64, wherein the rate of polysorbate hydrolysis is reduced by at least about 20%.

66. The formulated antibody partial composition according to any one of claims 57 to 65, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

67. A method for reducing the enzymatic hydrolysis rate of a composition obtained from a purification platform, wherein the sample is (a) A capture step including treatment by affinity chromatography, and (b) A purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography and multimodal chromatography. This includes subjecting the materials to the purification platform, which includes the materials in this order. The purification platform further comprises one or more deep filtration steps, The one or more deep filtration steps are performed before the capture step, after the capture step, or after the capture step and before the purification step, Each deep filtration step includes processing with a deep filter, The aforementioned deep filter, (i) Silica and polyacrylic fibers, (ii) Hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membrane, (iii) Cellulose fibers, diatomaceous earth, and perlite, A method comprising a material selected from the group consisting of the following, thereby reducing the enzymatic hydrolysis activity rate of the composition compared to the purification of the sample using the same purification platform that does not include the one or more deep filtration steps.

68. The method according to claim 67, wherein the enzyme hydrolysis activity rate is the enzyme polysorbate hydrolysis activity rate.

69. The method according to claim 67 or 68, wherein the relative decrease in the enzymatic hydrolysis activity rate of the composition is at least about 20% compared to the purification of the sample using the same purification platform without the deep filtration step.

70. A method for reducing the level of one or more hydrolytic enzymes in a composition obtained from a purification platform, wherein the sample is (a) A capture step including treatment by affinity chromatography, and (b) A purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography and multimodal chromatography. This includes subjecting the materials to the purification platform, which includes the materials in this order. The purification platform further comprises one or more deep filtration steps, The one or more deep filtration steps are performed before the capture step, after the capture step, and before the purification step, or after the purification step, Each deep filtration step includes processing with a deep filter, The aforementioned deep filter, (i) Silica and polyacrylic fibers, (ii) Hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membrane, (iii) Cellulose fibers, diatomaceous earth, and perlite, Includes a material selected from the group consisting of, A method for thereby reducing the level of one or more hydrolytic enzymes in the composition compared to the purification of the sample using the same purification platform that does not include the one or more deep filtration steps.

71. The method according to claim 70, wherein the one or more hydrolytic enzymes can hydrolyze the polysorbate.

72. The method according to claim 70 or 71, wherein the relative decrease in the level of the one or more hydrolytic enzymes in the composition is at least about 20% compared to the purification of the sample using the same purification platform without the deep filtration step.

73. A method for reducing the degradation of polysorbate in a composition obtained from a purification platform, comprising the step of providing a sample to the purification platform, wherein the purification platform processes the sample, (a) A capture step including treatment by affinity chromatography, and (b) A purification step comprising treatment by chromatography selected from the group consisting of HIC, cation exchange chromatography and multimodal chromatography. This includes subjecting the materials to the purification platform, which includes the materials in this order. The purification platform further comprises one or more deep filtration steps, The one or more deep filtration steps are performed before the capture step, after the capture step, or after the capture step and before the purification step, Each deep filtration step includes processing with a deep filter, The aforementioned deep filter, (i) Silica and polyacrylic fibers, (ii) Hydrogel Q (quaternary amine) functionalized nonwoven media and multizone microporous membrane, (iii) Cellulose fibers, diatomaceous earth, and perlite, Includes a material selected from the group consisting of, A method that thereby reduces the degradation of the polysorbate in the composition compared to the purification of the sample using the same purification platform that does not include one or more deep filtration steps.

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

75. The method according to any one of claims 67 to 74, wherein the deep filter comprising the silica and the polyacrylic fiber comprises a silica filter aid and polyacrylic fiber pulp.

76. The method according to any one of claims 67 to 74, wherein the deep filter comprising the hydrogel Q-functionalized nonwoven fabric medium and the multizone microporous membrane comprises four layers comprising the hydrogel Q-functionalized nonwoven fabric material and the nine-zone microporous membrane.

77. The method according to any one of claims 67 to 74, wherein the deep filter comprising cellulose fibers, diatomaceous earth, and perlite comprises two layers, each layer comprising a cellulose filter matrix, the cellulose filter matrix being impregnated with a filtration aid comprising one or more diatomaceous earth or perlite, and each layer further comprising a resin binder.

78. The method according to any one of claims 67 to 77, wherein the deep filter is selected based on the pH of the solution that enters the deep filter.

79. The method according to claim 78, wherein the deep filter containing the silica and the polyacrylic fiber is selected when the amount of the solution entering the deep filter is about 5 to about 6.

5.

80. The method according to claim 78, wherein the deep filter comprising the hydrogel Q-functionalized nonwoven fabric medium and the multizone microporous membrane is selected when the amount of solution entering the deep filter is about 7 to about 8.

5.

81. The method according to any one of claims 67 to 80, further comprising the step of selecting the deep filter based on the pH of the solution that enters the deep filter.

82. The method according to any one of claims 67 to 81, wherein the purification platform comprises, in this order, a deep filtration step including treatment with the deep filter comprising the hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane, a capture step including treatment by protein A chromatography, and the purification step.

83. The method according to claim 82, wherein the purification step includes treatment with HIC.

84. The method according to claim 83, wherein the HIC is phenyl SEPHAROSE® high-speed flow chromatography.

85. The method according to claim 82, wherein the purification step includes treatment by cation exchange chromatography.

86. The method according to claim 85, wherein the cation exchange chromatography is POROS® 50HS.

87. The method according to any one of claims 67 to 86, wherein the purification platform further comprises a second deep filtration step, which includes treatment with the deep filter containing the silica and the polyacrylic fibers, the second deep filtration step being performed after the capture step and before the purification step.

88. The method according to claim 82, wherein the purification step includes treatment by multimodal chromatography.

89. The method according to claim 88, wherein the multimodal chromatography is CaptoAdhere.

90. The method according to claim 88 or 89, wherein the purification platform further comprises a second deep filtration step comprising treatment with the deep filter comprising the hydrogel Q-functionalized nonwoven fabric medium and a multizone microporous membrane, the second deep filtration step being performed after the capture step and before the purification step.

91. The method according to any one of claims 67 to 90, wherein the purification platform is for purifying a target from the sample, and the sample comprises the target and one or more host cell impurities.

92. The method according to claim 91, wherein the target comprises a polypeptide.

93. The method according to claim 91 or 92, wherein the host cell impurity is a host cell protein.

94. The method according to any one of claims 67 to 93, wherein the purification platform further comprises a virus inactivation step, the virus inactivation step being performed after the capture step.

95. The method according to claim 94, wherein one or more deep filtration steps are performed after the virus inactivation step.

96. The method according to any one of claims 67 to 95, wherein the purification platform further comprises an ultrafiltration / diafiltration (UFDF) step, the UFDF step being performed after the purification step.

97. The method according to any one of claims 67 to 96, further comprising determining the enzyme hydrolysis activity rate of the composition.

98. The method according to any one of claims 67 to 97, further comprising determining the level of one or more hydrolytic enzymes in the composition.

99. The method according to any one of claims 67 to 98, wherein the composition comprises polysorbate.

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

101. The method according to any one of claims 67 to 100, further comprising a sample processing step.

102. The method according to any one of claims 67 to 101, wherein the sample is a cell culture sample or derived from a cell culture sample.

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

104. The method according to any one of claims 67 to 103, wherein the sample comprises a host cell or a component derived therefrom.

105. The method according to any one of claims 67 to 104, wherein the sample comprises one or more host cell proteins, and one of the one or more host cell proteins is a hydrolase.

106. The method according to claim 105, wherein the hydrolytic enzyme is lipase, esterase, thioesterase, phospholipase, or ceramidase.

107. The method according to any one of claims 67 to 106, wherein the sample comprises a target, and the target is an antibody portion.

108. The method according to claim 107, wherein the antibody portion is a monoclonal antibody.

109. The method according to claim 107 or 108, wherein the antibody portion is a human antibody, a humanized antibody, or a chimeric antibody.

110. The method according to any one of claims 107 to 109, wherein the antibody portion is selected from the group consisting of anti-CD20 antibody, anti-CD40 antibody, anti-HER2 antibody, anti-IL6 antibody, anti-IgE antibody, anti-IL13 antibody, anti-TIGIT antibody, anti-PD-L1 antibody, anti-VEGF-A antibody, anti-VEGF-A / ANG2 antibody, anti-CD79b antibody, anti-ST2 antibody, anti-factor D antibody, anti-factor IX antibody, anti-factor X antibody, anti-α beta antibody, anti-tau antibody, anti-CEA antibody, anti-CEA / CD3 antibody, anti-CD20 / CD3 antibody, anti-FcRH5 / CD3 antibody, anti-HER2 / CD3 antibody, anti-FFFR1 / KLB antibody, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, and TYRP1 TCB antibody.

111. The method according to any one of claims 107 to 110, wherein the antibody portion is selected from the group consisting of ocrelizumab, pertuzumab, trastuzumab, tocilizumab, falisimab, polatuzumab, gantenerumab, sibisatamab, crenezumab, mosnetuzumab, tilagolmab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lamparizumab, lebrikizumab, omalizumab, ranibizumab, emicizumab, sericrelumab, pracinezumab, RO6874281, and RO7122290.

112. A pharmaceutical composition obtained by the method according to any one of claims 67 to 111.

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