Mitigating Therapeutic Protein Fragmentation During the Cell Culture Harvest Process

Abstract

Disclosed herein is a method for recovering antibodies as recombinant proteins containing an asparaginyl endopeptidase (AEP, legumain) cleavage site with amino acids NF by maintaining a post-fermentation pH between 4.6 and 5.3 for acidification to precipitate impurities to prevent self-cleavage of the proenzyme and therefore activation of the peptidase at lower pH, and a method for inhibiting cleavage of recombinant proteins containing the AEP cleavage site during the biomanufacturing process.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 482,844, filed February 2, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] Submission of sequence listing This application contains a computer-readable Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy (created on January 12, 2024) is titled "10177-WO01-SEC_ST26" and is 4,935 bytes in size.

[0003] The present disclosure relates to a method for recovering a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site, the method comprising: an acid precipitation procedure at a pH of about 4.6 to about 5.3; and, optionally, (i) employing a dissolved oxygen level of about 64 mmHg to about 128 mmHg during cell culture cool-down and / or harvest; and / or (ii) utilizing an exogenous protease inhibitor, e.g., during one or more of the cell culture growth phase, cell culture production phase, and / or cell culture cool-down. The present disclosure also relates to a method for inhibiting cleavage of a recombinant protein containing an AEP cleavage site during a biomanufacturing process involving an acid precipitation procedure, wherein if one or more parameters (e.g., fragment amount; AEP amount or activity) exceed a threshold value, the acid precipitation pH is adjusted to about 4.6 to about 5.3. Furthermore, the present disclosure relates to a method for monitoring the activation of AEP in a sample derived from a biomanufacturing process utilizing Chinese hamster ovary (CHO) cells, the method comprising subjecting the sample to trypsin to obtain a trypsin-digested sample, and measuring the amount of LMSTNDLK (SEQ ID NO: 1) and / or LDLTPSPEVPTILK (SEQ ID NO: 2) in the trypsin-digested sample. [Background technology]

[0004] In response to the intense and growing demand for biotherapeutics, significant advances in bioprocessing have been made, facilitating cost-effective, large-scale recombinant protein production. For example, new and improved high cell density cultivation methods and intensified cell culture processes have enabled greater productivity while reducing costs. However, in addition to increasing protein titer, these upstream processes typically result in higher cell densities and process-related impurity levels (e.g., host cell proteins (HCPs) and nucleic acids), which increase the burden on costly downstream clarification and purification operations used to isolate recombinant proteins.

[0005] Cell culture clarification is a downstream unit operation in which cells, cell debris, and other process-related impurities are removed from cell culture harvest fluid prior to further downstream purification steps, such as chromatographic separation processes. Mechanical separation (e.g., centrifugation) followed by depth filtration is a common approach for clarifying cell cultures. To increase filtration throughput and improve separation performance, many clarification processes incorporate pretreatment steps (e.g., acid precipitation or addition of flocculants) to insolubilize certain process-related impurities before mechanically separating the remaining cell culture fluid (CCF).

[0006] However, as with all downstream unit operations, the process conditions used during cell harvesting must be carefully controlled to avoid product loss and degradation. This is especially true when processing large-scale cell biomass due to the high concentrations of lysosomal enzymes (e.g., cathepsins and pepsins) that can be secreted or released into the bioreactor during cell cultivation and harvesting. While lysosomal enzymes are not active under the neutral pH conditions employed during cell culture, the enzymes can become active under acidic and slightly acidic pH conditions such as those used during cell culture harvest and protein purification. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need in the art for new and improved cell culture harvest methods that allow for efficient clarification of high cell density cultures without inducing product cleavage by lysosomal enzymes that may be present in the cell culture. [Means for solving the problem]

[0008] Disclosed herein is a method for recovering a recombinant protein during a biomanufacturing process, comprising: mixing the acidic solution with the cell culture containing the recombinant protein to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and incubating the acidified cell culture to induce aggregation of one or more cell culture impurities; Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. It is a method.

[0009] In some embodiments, the cell culture is mixed with an exogenous protease inhibitor prior to mixing with the acidic solution. In some embodiments, the exogenous protease inhibitor is mixed with the acidified cell culture. In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0010] In some embodiments, an exogenous protease inhibitor is added to a downstream pool of the acidified cell culture (e.g., the Protein A pool or the low pH viral inactivation pool). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0011] In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 4° C. to about 37° C. In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 15° C. to about 20° C.

[0012] In some embodiments, the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing. In some embodiments, the acidic solution comprises an acid selected from acetic acid, phosphoric acid, and combinations thereof. In some embodiments, the acidic solution comprises sulfuric acid. In some embodiments, the acidic solution comprises citric acid. In some embodiments, the acidic solution comprises acetic acid. In some embodiments, the acidic solution comprises 1 M acetic acid. In some embodiments, the acidic solution comprises phosphoric acid. In some embodiments, the acidic solution comprises 2 M phosphoric acid.

[0013] In some embodiments, the method comprises incubating the acidified cell culture for at least about 30 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 40 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 45 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 60 minutes.

[0014] In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 48 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 24 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 12 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 120 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 90 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 60 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 30 minutes to about 90 minutes.

[0015] In some embodiments, the method further comprises separating one or more cell culture impurities from the acidified cell culture to obtain a clarified cell culture. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation, disc stack centrifugation, and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by disc stack centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by intermittent discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration without centrifugation, hi some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation and depth filtration.

[0016] In some embodiments, the depth filtration is performed at a rate of about 150 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 140 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 130 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 120 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 110 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 100 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 95 L / m 2In some embodiments, the depth filtration is performed at a load of less than about 90 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 85 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 80 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 75 L / m 2 Perform with a loading dose of less than

[0017] In some embodiments, the method includes neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture. In some embodiments, the method does not include neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture.

[0018] In some embodiments, the method includes neutralizing the clarified cell culture.

[0019] In some embodiments, the one or more cell culture impurities comprise cell culture debris, host cell proteins, and nucleic acids. In some embodiments, the one or more cell culture impurities comprise cell culture debris. In some embodiments, the one or more cell culture impurities comprise host cell proteins. In some embodiments, the one or more cell culture impurities comprise nucleic acids. In some embodiments, the one or more cell culture impurities are selected from host cell proteins and nucleic acids.

[0020] In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.2.

[0021] In some embodiments, the pH of the acidified cell culture is about 4.6. In some embodiments, the pH of the acidified cell culture is about 4.7. In some embodiments, the pH of the acidified cell culture is about 4.8. In some embodiments, the pH of the acidified cell culture is about 4.9. In some embodiments, the pH of the acidified cell culture is about 5.0. In some embodiments, the pH of the acidified cell culture is about 5.1. In some embodiments, the pH of the acidified cell culture is about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.3.

[0022] In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.2 pH units during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.1 pH units during incubation.

[0023] In some embodiments, the pH of the acidified cell culture deviates by less than about 25% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 20% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 15% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 10% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 5% during incubation.

[0024] In some embodiments, the acidified cell culture comprises a recombinant protein comprising a sequence upstream of an AEP cleavage site and a sequence downstream of the AEP cleavage site, hi some embodiments, the acidified cell culture comprises a recombinant protein comprising an antibody heavy chain sequence upstream of an AEP cleavage site and an antibody heavy chain sequence downstream of the AEP cleavage site.

[0025] In some embodiments, the acidified cell culture has a reduced amount of one or more fragments of the recombinant protein compared to an alternative acidified cell culture having a pH of less than about 4.6, hi some embodiments, the fragments of the recombinant protein include species that are cleaved at an AEP cleavage site.

[0026] In some embodiments, less than about 5 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 4 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 3 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 2 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 1 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. Fragment content can be assessed, for example, by UV absorbance relative peak area by reduced capillary electrophoresis.

[0027] In some embodiments, the method further comprises measuring the amount or activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP prior to mixing. In some embodiments, the method further comprises measuring the activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP proenzyme prior to mixing.

[0028] In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry or an immunoassay. In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry. In some embodiments, the amount of AEP or AEP proenzyme is measured using an immunoassay.

[0029] In some embodiments, the biomanufacturing process utilizes CHO cells, and the amount of AEP proenzyme is measured by subjecting the pooled sample to trypsin to obtain a trypsin-digested sample and measuring the amount of LMSTNDLK (SEQ ID NO: 1) and / or LDLTPSPEVLTILK (SEQ ID NO: 2) in the trypsin-digested sample. In some embodiments, the method further includes comparing the measured amount of SEQ ID NO: 1 and / or SEQ ID NO: 2 to one or more benchmark values, such as, for example, the measured amount in a trypsin-digested sample containing a known amount of AEP at a pH of 5.5 or greater.

[0030] In some embodiments, the activity of AEP is measured using a cleavage assay.

[0031] In some embodiments, the cell culture comprises mammalian cells, hi some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells.

[0032] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0033] In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0034] In some embodiments, the biomanufacturing process comprises a perfusion cell culture process, an enriched fed-batch cell culture process, or an intensive cell culture process. In some embodiments, the biomanufacturing process comprises a perfusion cell culture process. In some embodiments, the biomanufacturing process comprises an enriched fed-batch cell culture process. In some embodiments, the biomanufacturing process comprises an intensive cell culture process.

[0035] In some embodiments, the biomanufacturing process is carried out to produce a viable cell density of at least about 10 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 5 x 10 cells / mL. 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 10 cells / mL. 7 Contains the production phase, which is 100 cells / mL.

[0036] In some embodiments, the biomanufacturing process includes a production phase with a packed cell volume of about 2% to about 40%. In some embodiments, the biomanufacturing process includes a production phase with a packed cell volume of about 15% to about 18%. In some embodiments, the biomanufacturing process includes a production phase with a packed cell volume of about 25% to about 35%. In some embodiments, the biomanufacturing process includes a production phase with a packed cell volume of about 3% to about 15%.

[0037] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 20 kL.

[0038] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 20 kL.

[0039] Also disclosed herein is a method for recovering a recombinant protein during a biomanufacturing process, comprising: establishing a cell culture by inoculating a bioreactor with mammalian cells expressing the recombinant protein; Maintaining cell cultures during growth and production phases; cooling the cell culture, wherein the cell culture is cooled to a dissolved oxygen level of about 64 mmHg to about 128 mmHg; mixing the cell culture with an acidic solution to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and Incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. It is a method.

[0040] In some embodiments, the cell culture is cooled to about 4°C to about 15°C. In some embodiments, the cell culture is cooled to about 4°C to about 10°C. In some embodiments, the cell culture is cooled to about 9°C to about 11°C. In some embodiments, the cell culture is cooled to about 10°C.

[0041] In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 4° C. to about 37° C. In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 15° C. to about 20° C.

[0042] In some embodiments, the cell culture is combined with exogenous protease inhibitors during the growth phase, production phase, and / or cooling phase. In some embodiments, the cell culture is combined with exogenous protease inhibitors after the production phase. In some embodiments, the cell culture is combined with exogenous protease inhibitors after the production phase and before the cooling phase.

[0043] In some embodiments, an exogenous protease inhibitor is mixed with the cell culture prior to mixing with the acidic solution. In some embodiments, an exogenous protease inhibitor is mixed with the acidified cell culture. In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0044] In some embodiments, an exogenous protease inhibitor is added to a downstream pool of the acidified cell culture (e.g., the Protein A pool or the low pH viral inactivation pool). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0045] In some embodiments, the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing. In some embodiments, the acidic solution comprises an acid selected from acetic acid, phosphoric acid, and combinations thereof. In some embodiments, the acidic solution comprises sulfuric acid. In some embodiments, the acidic solution comprises citric acid. In some embodiments, the acidic solution comprises acetic acid. In some embodiments, the acidic solution comprises 1 M acetic acid. In some embodiments, the acidic solution comprises phosphoric acid. In some embodiments, the acidic solution comprises 2 M phosphoric acid.

[0046] In some embodiments, the method comprises incubating the acidified cell culture for at least about 30 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 40 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 45 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 60 minutes.

[0047] In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 48 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 24 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 12 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 120 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 90 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 60 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 30 minutes to about 90 minutes.

[0048] In some embodiments, the method further comprises separating one or more cell culture impurities from the acidified cell culture to obtain a clarified cell culture. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation, disc stack centrifugation, and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by disc stack centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by intermittent discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration without centrifugation, hi some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation and depth filtration.

[0049] In some embodiments, the depth filtration is performed at a rate of about 150 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 140 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 130 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 120 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 110 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 100 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 95 L / m2 In some embodiments, the depth filtration is performed at a load of less than about 90 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 85 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 80 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 75 L / m 2 Perform with a loading dose of less than

[0050] In some embodiments, the method includes neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture. In some embodiments, the method does not include neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture.

[0051] In some embodiments, the method includes neutralizing the clarified cell culture.

[0052] In some embodiments, the one or more cell culture impurities comprise cell culture debris, host cell proteins, and nucleic acids. In some embodiments, the one or more cell culture impurities comprise cell culture debris. In some embodiments, the one or more cell culture impurities comprise host cell proteins. In some embodiments, the one or more cell culture impurities comprise nucleic acids. In some embodiments, the one or more cell culture impurities are selected from host cell proteins and nucleic acids.

[0053] In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.2.

[0054] In some embodiments, the pH of the acidified cell culture is about 4.6. In some embodiments, the pH of the acidified cell culture is about 4.7. In some embodiments, the pH of the acidified cell culture is about 4.8. In some embodiments, the pH of the acidified cell culture is about 4.9. In some embodiments, the pH of the acidified cell culture is about 5.0. In some embodiments, the pH of the acidified cell culture is about 5.1. In some embodiments, the pH of the acidified cell culture is about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.3.

[0055] In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.2 pH units during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.1 pH units during incubation.

[0056] In some embodiments, the pH of the acidified cell culture deviates by less than about 25% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 20% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 15% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 10% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 5% during incubation.

[0057] In some embodiments, the acidified cell culture comprises a recombinant protein comprising a sequence upstream of an AEP cleavage site and a sequence downstream of the AEP cleavage site.

[0058] In some embodiments, the acidified cell culture has a reduced amount of one or more fragments of the recombinant protein compared to an alternative acidified cell culture having a pH of less than about 4.6, hi some embodiments, the fragments of the recombinant protein include species that are cleaved at an AEP cleavage site.

[0059] In some embodiments, less than about 5 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 4 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 3 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 2 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 1 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. Fragment content can be assessed, for example, by UV absorbance relative peak area by reduced capillary electrophoresis.

[0060] In some embodiments, the method further comprises measuring the amount or activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP prior to mixing. In some embodiments, the method further comprises measuring the activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP proenzyme prior to mixing.

[0061] In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry or an immunoassay. In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry. In some embodiments, the amount of AEP or AEP proenzyme is measured using an immunoassay.

[0062] In some embodiments, the biomanufacturing process utilizes CHO cells, and the amount of AEP proenzyme is measured by subjecting the pooled sample to trypsin to obtain a trypsin-digested sample and measuring the amount of LMSTNDLK (SEQ ID NO: 1) and / or LDLTPSPEVLTILK (SEQ ID NO: 2) in the trypsin-digested sample. In some embodiments, the method further includes comparing the measured amount of SEQ ID NO: 1 and / or SEQ ID NO: 2 to one or more benchmark values, such as, for example, the measured amount in a trypsin-digested sample containing a known amount of AEP at a pH of 5.5 or greater.

[0063] In some embodiments, the activity of AEP is measured using a cleavage assay.

[0064] In some embodiments, the mammalian cell is a CHO cell.

[0065] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0066] In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0067] In some embodiments, the biomanufacturing process comprises a perfusion cell culture process, an enriched fed-batch cell culture process, or an intensive cell culture process. In some embodiments, the biomanufacturing process comprises a perfusion cell culture process. In some embodiments, the biomanufacturing process comprises an enriched fed-batch cell culture process. In some embodiments, the biomanufacturing process comprises an intensive cell culture process.

[0068] In some embodiments, the biomanufacturing process is carried out to produce a viable cell density of at least about 10 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 5 x 10 cells / mL. 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 10 cells / mL. 7 Contains the production phase, which is 100 cells / mL.

[0069] In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 2% to about 40%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 15% to about 18%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 25% to about 35%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 3% to about 15%.

[0070] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 20 kL.

[0071] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 20 kL.

[0072] Also disclosed herein is a method for recovering a recombinant protein during a biomanufacturing process, comprising: establishing a cell culture by inoculating a bioreactor with mammalian cells expressing the recombinant protein; maintaining the cell culture during a growth phase and a production phase, wherein the cell culture is mixed with an exogenous protease inhibitor during the growth phase and / or during or after the production phase; combining the cell culture with an acidic solution to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. It is a method.

[0073] In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0074] In some embodiments, the cell culture is mixed with an exogenous protease inhibitor prior to mixing with the acidic solution. In some embodiments, the exogenous protease inhibitor is mixed with the acidified cell culture. In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0075] In some embodiments, an exogenous protease inhibitor is added to a downstream pool of the acidified cell culture (e.g., the Protein A pool or the low pH viral inactivation pool). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0076] In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 4° C. to about 37° C. In some embodiments, the method comprises incubating the acidified cell culture at a temperature of about 15° C. to about 20° C.

[0077] In some embodiments, the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing. In some embodiments, the acidic solution comprises an acid selected from acetic acid, phosphoric acid, and combinations thereof. In some embodiments, the acidic solution comprises sulfuric acid. In some embodiments, the acidic solution comprises citric acid. In some embodiments, the acidic solution comprises acetic acid. In some embodiments, the acidic solution comprises 1 M acetic acid. In some embodiments, the acidic solution comprises phosphoric acid. In some embodiments, the acidic solution comprises 2 M phosphoric acid.

[0078] In some embodiments, the method comprises incubating the acidified cell culture for at least about 30 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 40 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 45 minutes. In some embodiments, the method comprises incubating the acidified cell culture for at least about 60 minutes.

[0079] In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 48 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 24 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 12 hours. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 120 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 90 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 5 minutes to about 60 minutes. In some embodiments, the method comprises incubating the acidified cell culture for about 30 minutes to about 90 minutes.

[0080] In some embodiments, the method further comprises separating one or more cell culture impurities from the acidified cell culture to obtain a clarified cell culture. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation, disc stack centrifugation, and / or depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by disc stack centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by intermittent discharge centrifugation. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration. In some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by depth filtration without centrifugation, hi some embodiments, the one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation and depth filtration.

[0081] In some embodiments, the depth filtration is performed at a rate of about 150 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 140 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 130 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 120 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 110 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 100 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 95 L / m 2In some embodiments, the depth filtration is performed at a load of less than about 90 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 85 L / m 2 In some embodiments, the depth filtration is performed at a load of less than about 80 L / m 2 In some embodiments, the depth filtration is performed at a loading of less than about 75 L / m 2 Perform with a loading dose of less than

[0082] In some embodiments, the method includes neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture. In some embodiments, the method does not include neutralizing the acidified cell culture prior to separating the one or more cell culture impurities from the acidified cell culture.

[0083] In some embodiments, the method includes neutralizing the clarified cell culture.

[0084] In some embodiments, the one or more cell culture impurities comprise cell culture debris, host cell proteins, and nucleic acids. In some embodiments, the one or more cell culture impurities comprise cell culture debris. In some embodiments, the one or more cell culture impurities comprise host cell proteins. In some embodiments, the one or more cell culture impurities comprise nucleic acids. In some embodiments, the one or more cell culture impurities are selected from host cell proteins and nucleic acids.

[0085] In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.3. In some embodiments, the pH of the acidified cell culture is about 4.7 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.8 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 4.9 to about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.0 to about 5.2.

[0086] In some embodiments, the pH of the acidified cell culture is about 4.6. In some embodiments, the pH of the acidified cell culture is about 4.7. In some embodiments, the pH of the acidified cell culture is about 4.8. In some embodiments, the pH of the acidified cell culture is about 4.9. In some embodiments, the pH of the acidified cell culture is about 5.0. In some embodiments, the pH of the acidified cell culture is about 5.1. In some embodiments, the pH of the acidified cell culture is about 5.2. In some embodiments, the pH of the acidified cell culture is about 5.3.

[0087] In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.2 pH units during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about ±0.1 pH units during incubation.

[0088] In some embodiments, the pH of the acidified cell culture deviates by less than about 25% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 20% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 15% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 10% during incubation. In some embodiments, the pH of the acidified cell culture deviates by less than about 5% during incubation.

[0089] In some embodiments, the acidified cell culture comprises a recombinant protein comprising a sequence upstream of an AEP cleavage site and a sequence downstream of the AEP cleavage site.

[0090] In some embodiments, the acidified cell culture has a reduced amount of one or more fragments of the recombinant protein compared to an alternative acidified cell culture having a pH of less than about 4.6, hi some embodiments, the fragments of the recombinant protein include species that are cleaved at an AEP cleavage site.

[0091] In some embodiments, less than about 5 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 4 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 3 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 2 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. In some embodiments, less than about 1 wt% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein. Fragment content can be assessed, for example, by UV absorbance relative peak area by reduced capillary electrophoresis.

[0092] In some embodiments, the method further comprises measuring the amount or activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP prior to mixing. In some embodiments, the method further comprises measuring the activity of AEP prior to mixing. In some embodiments, the method further comprises measuring the amount of AEP proenzyme prior to mixing.

[0093] In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry or an immunoassay. In some embodiments, the amount of AEP or AEP proenzyme is measured using mass spectrometry. In some embodiments, the amount of AEP or AEP proenzyme is measured using an immunoassay.

[0094] In some embodiments, the mammalian cells are CHO cells, and the amount of AEP proenzyme is measured by subjecting the pooled samples to trypsin to obtain a trypsin-digested sample and measuring the amount of LMSTNDLK (SEQ ID NO: 1) and / or LDLTPSPEVLTILK (SEQ ID NO: 2) in the trypsin-digested sample. In some embodiments, the method further includes comparing the measured amount of SEQ ID NO: 1 and / or SEQ ID NO: 2 to one or more benchmark values, such as, for example, the measured amount in a trypsin-digested sample containing a known amount of AEP at a pH of 5.5 or greater.

[0095] In some embodiments, the activity of AEP is measured using a cleavage assay.

[0096] In some embodiments, the mammalian cell is a CHO cell.

[0097] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0098] In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0099] In some embodiments, the biomanufacturing process comprises a perfusion cell culture process, an enriched fed-batch cell culture process, or an intensive cell culture process. In some embodiments, the biomanufacturing process comprises a perfusion cell culture process. In some embodiments, the biomanufacturing process comprises an enriched fed-batch cell culture process. In some embodiments, the biomanufacturing process comprises an intensive cell culture process.

[0100] In some embodiments, the biomanufacturing process is carried out to produce a viable cell density of at least about 10 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 5 x 10 cells / mL. 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 10 cells / mL. 7 Contains the production phase, which is 100 cells / mL.

[0101] In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 2% to about 40%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 15% to about 18%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 25% to about 35%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 3% to about 15%.

[0102] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 20 kL.

[0103] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 20 kL.

[0104] Also disclosed herein is a method for inhibiting cleavage of a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site during a biomanufacturing process involving acid precipitation, the method comprising: Measuring the amount or activity of AEP in a sample isolated from the cell culture, harvest, or purification operations of the biomanufacturing process; and If the amount or activity of AEP exceeds a threshold, the pH of the acid precipitate is adjusted to about 4.6 to about 5.3. The method includes:

[0105] In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.7 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.8 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.9 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 5.0 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.7 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.8 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.9 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 5.0 to about 5.2.

[0106] In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.6. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.7. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.8. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.9. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.0. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.1. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.2. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.3.

[0107] In some embodiments, adjusting the pH of the acid precipitation comprises mixing the cell culture with an acidic solution. In some embodiments, the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing. In some embodiments, the acidic solution comprises an acid selected from acetic acid, phosphoric acid, and combinations thereof. In some embodiments, the acidic solution comprises sulfuric acid. In some embodiments, the acidic solution comprises citric acid. In some embodiments, the acidic solution comprises acetic acid. In some embodiments, the acidic solution comprises 1 M acetic acid. In some embodiments, the acidic solution comprises phosphoric acid. In some embodiments, the acidic solution comprises 2 M phosphoric acid.

[0108] In some embodiments, acid precipitation is carried out for at least about 30 minutes. In some embodiments, acid precipitation is carried out for at least about 40 minutes. In some embodiments, acid precipitation is carried out for at least about 45 minutes. In some embodiments, acid precipitation is carried out for at least about 60 minutes.

[0109] In some embodiments, acid precipitation is carried out for about 5 minutes to about 24 hours. In some embodiments, acid precipitation is carried out for about 5 minutes to about 12 hours. In some embodiments, acid precipitation is carried out for about 5 minutes to about 120 minutes. In some embodiments, acid precipitation is carried out for about 5 minutes to about 90 minutes. In some embodiments, acid precipitation is carried out for about 5 minutes to about 60 minutes. In some embodiments, acid precipitation is carried out for about 30 minutes to about 90 minutes.

[0110] In some embodiments, cleavage of the recombinant protein is inhibited compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 25% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 50% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 75% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6.

[0111] In some embodiments, the method includes measuring the amount of AEP. In some embodiments, the amount of AEP is measured using mass spectrometry or an immunoassay. In some embodiments, the amount of AEP is measured using mass spectrometry. In some embodiments, the amount of AEP is measured using an immunoassay.

[0112] In some embodiments, the AEP amount threshold is greater than about 500 ng AEP per mg of harvest cell culture fluid (HCCF). In some embodiments, the AEP amount threshold is greater than about 750 ng AEP per mg of HCCF. In some embodiments, the AEP amount threshold is greater than about 1000 ng AEP per mg of HCCF. In some embodiments, the AEP amount threshold is greater than about 1500 ng AEP per mg of HCCF. In some embodiments, the AEP amount threshold is greater than about 2000 ng AEP per mg of HCCF.

[0113] In some embodiments, the threshold amount of AEP is about 500 ng of AEP per mg of HCCF. In some embodiments, the threshold amount of AEP is about 750 ng of AEP per mg of HCCF. In some embodiments, the threshold amount of AEP is about 1000 ng of AEP per mg of HCCF. In some embodiments, the threshold amount of AEP is about 1500 ng of AEP per mg of HCCF. In some embodiments, the threshold amount of AEP is about 2000 ng of AEP per mg of HCCF.

[0114] In some embodiments, the method includes measuring the activity of AEP. In some embodiments, the activity of AEP is measured using a cleavage assay.

[0115] In some embodiments, the threshold activity of AEP is greater than about 1% clipping of the recombinant protein at the AEP cleavage site. In some embodiments, the threshold activity of AEP is greater than about 2% clipping of the recombinant protein at the AEP cleavage site. In some embodiments, the threshold activity of AEP is greater than about 3% clipping of the recombinant protein at the AEP cleavage site. In some embodiments, the threshold activity of AEP is greater than about 4% clipping of the recombinant protein at the AEP cleavage site. In some embodiments, the threshold activity of AEP is greater than about 5% clipping of the recombinant protein at the AEP cleavage site.

[0116] In some embodiments, the sample is isolated from a cell culture operation or a harvesting operation. In some embodiments, the sample is isolated from a cell culture operation. In some embodiments, the sample is isolated from a harvesting operation.

[0117] In some embodiments, the method further includes adjusting the dissolved oxygen level of the cell culture to between about 64 mmHg and about 128 mmHg during a cell culture cooling operation of the biomanufacturing process if the amount or activity of the AEP exceeds a threshold value.

[0118] In some embodiments, the method further comprises combining an exogenous protease inhibitor with the cell culture during the growth and / or production phase of the biomanufacturing process if the amount or activity of AEP is above a threshold value, hi some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0119] In some embodiments, an exogenous protease inhibitor is added to a downstream pool of the acidified cell culture (e.g., the Protein A pool or the low pH viral inactivation pool). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0120] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0121] In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0122] In some embodiments, the biomanufacturing process comprises a cell culture process selected from a perfusion cell culture process, an enriched fed-batch cell culture process, and an enriched cell culture process, wherein the cell culture process utilizes mammalian cells. In some embodiments, the mammalian cells are selected from CHO cells. In some embodiments, the biomanufacturing process comprises a perfusion cell culture process. In some embodiments, the biomanufacturing process comprises an enriched fed-batch cell culture process. In some embodiments, the biomanufacturing process comprises an enriched cell culture process.

[0123] In some embodiments, the biomanufacturing process is carried out to produce a viable cell density of at least about 10 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 5 x 10 cells / mL. 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 10 cells / mL. 7 Contains the production phase, which is 100 cells / mL.

[0124] In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 2% to about 40%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 15% to about 18%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 25% to about 35%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 3% to about 15%.

[0125] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 20 kL.

[0126] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 20 kL.

[0127] Also disclosed herein is a method for inhibiting cleavage of a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site during a biomanufacturing process involving acid precipitation, the method comprising: measuring the amount of one or more fragments of the recombinant protein in a sample isolated during a cell culture, harvest, or purification operation of the biomanufacturing process; and If the amount of one or more fragments exceeds a threshold value, the pH of the acid precipitation is adjusted to about 4.6 to about 5.3. The method includes:

[0128] In some embodiments, the threshold is at least about 1% w / w of the recombinant protein being one or more fragments. In some embodiments, the threshold is at least about 2% w / w of the recombinant protein being one or more fragments. In some embodiments, the threshold is at least about 3% w / w of the recombinant protein being one or more fragments. In some embodiments, the threshold is at least about 4% w / w of the recombinant protein being one or more fragments. In some embodiments, the threshold is at least about 5% w / w of the recombinant protein being one or more fragments. Fragment content may be assessed, for example, by UV absorbance relative peak area by reduced capillary electrophoresis.

[0129] In some embodiments, the one or more fragments include a species that is cleaved at an AEP cleavage site.

[0130] In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.7 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.8 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.9 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 5.0 to about 5.3. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.7 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.8 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 4.9 to about 5.2. In some embodiments, the method comprises adjusting the pH of the acid precipitation to about 5.0 to about 5.2.

[0131] In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.6. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.7. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.8. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 4.9. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.0. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.1. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.2. In some embodiments, the method includes adjusting the pH of the acid precipitation to about 5.3.

[0132] In some embodiments, adjusting the pH of the acid precipitation comprises mixing the cell culture with an acidic solution. In some embodiments, the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing. In some embodiments, the acidic solution comprises an acid selected from acetic acid, phosphoric acid, and combinations thereof. In some embodiments, the acidic solution comprises sulfuric acid. In some embodiments, the acidic solution comprises citric acid. In some embodiments, the acidic solution comprises acetic acid. In some embodiments, the acidic solution comprises 1 M acetic acid. In some embodiments, the acidic solution comprises phosphoric acid. In some embodiments, the acidic solution comprises 2 M phosphoric acid.

[0133] In some embodiments, acid precipitation is carried out for at least about 30 minutes. In some embodiments, acid precipitation is carried out for at least about 40 minutes. In some embodiments, acid precipitation is carried out for at least about 45 minutes. In some embodiments, acid precipitation is carried out for at least about 60 minutes.

[0134] In some embodiments, acid precipitation is carried out for about 5 minutes to about 24 hours. In some embodiments, acid precipitation is carried out for about 5 minutes to about 12 hours. In some embodiments, acid precipitation is carried out for about 5 minutes to about 120 minutes. In some embodiments, acid precipitation is carried out for about 5 minutes to about 90 minutes. In some embodiments, acid precipitation is carried out for about 5 minutes to about 60 minutes. In some embodiments, acid precipitation is carried out for about 30 minutes to about 90 minutes.

[0135] In some embodiments, cleavage of the recombinant protein is inhibited compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 25% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 50% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6. In some embodiments, cleavage of the recombinant protein is inhibited by at least about 75% compared to a recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6.

[0136] In some embodiments, the sample is isolated from a cell culture operation or a harvesting operation. In some embodiments, the sample is isolated from a cell culture operation. In some embodiments, the sample is isolated from a harvesting operation.

[0137] In some embodiments, the method further includes adjusting the dissolved oxygen level of the cell culture to between about 64 mmHg and about 128 mmHg during a cell culture cooling operation of the biomanufacturing process if the amount of one or more fragments exceeds a threshold value.

[0138] In some embodiments, the method further comprises combining an exogenous protease inhibitor with the cell culture during the growth and / or production phase of the biomanufacturing process if the amount of one or more fragments exceeds a threshold value, hi some embodiments, the exogenous protease inhibitor is an AEP inhibitor.

[0139] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0140] In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0141] In some embodiments, the biomanufacturing process comprises a cell culture process selected from a perfusion cell culture process, an enriched fed-batch cell culture process, and an enriched cell culture process, wherein the cell culture process utilizes mammalian cells. In some embodiments, the mammalian cells are selected from CHO cells. In some embodiments, the biomanufacturing process comprises a perfusion cell culture process. In some embodiments, the biomanufacturing process comprises an enriched fed-batch cell culture process. In some embodiments, the biomanufacturing process comprises an enriched cell culture process.

[0142] In some embodiments, the biomanufacturing process is carried out to produce a viable cell density of at least about 10 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 5 x 10 cells / mL. 6 In some embodiments, the biomanufacturing process includes a production phase in which the viable cell density is at least about 10 cells / mL. 7 Contains the production phase, which is 100 cells / mL.

[0143] In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 2% to about 40%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 15% to about 18%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 25% to about 35%. In some embodiments, the biomanufacturing process includes a production phase with a compacted cell volume of about 3% to about 15%.

[0144] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 20 kL.

[0145] In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 500 L. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 1 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 2 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 5 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 10 kL. In some embodiments, the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of about 20 kL.

[0146] Also disclosed herein is a method for monitoring the activation of AEP in a sample from a biomanufacturing process utilizing CHO cells, the method comprising subjecting the sample to trypsin to obtain a trypsin-digested sample and measuring the amount of LMSTNDLK (SEQ ID NO: 1) and / or LDLTPSPEVLTILK (SEQ ID NO: 2) in the trypsin-digested sample. In some embodiments, the method further comprises comparing the measured amount of SEQ ID NO: 1 and / or SEQ ID NO: 2 to one or more benchmark values, such as, for example, the measured amount in a trypsin-digested sample containing a known amount of AEP at a pH of 5.5 or greater.

[0147] Non-limiting exemplary features Some exemplary embodiments / features of the present disclosure include, but are not limited to:

[0148] E1. A method for recovering a recombinant protein during a biomanufacturing process, comprising: mixing the acidic solution with the cell culture containing the recombinant protein to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and Incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. method.

[0149] E2. A method for recovering a recombinant protein during a biomanufacturing process, comprising: establishing a cell culture by inoculating a bioreactor with mammalian cells expressing the recombinant protein; Maintaining cell cultures during growth and production phases; cooling the cell culture, wherein the cell culture is cooled to a dissolved oxygen level of about 64 mmHg to about 128 mmHg; mixing the cell culture with an acidic solution to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and Incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. method.

[0150] E3. The method according to E2, wherein the cell culture is cooled to about 4°C to about 15°C (for example, about 9°C to about 11°C).

[0151] E4. The method of E2 or E3, wherein the cell culture is cooled to about 10°C.

[0152] E5. The method of any one of E2-E4, wherein the cell culture is admixed with an exogenous protease inhibitor during the growth phase, production phase, and / or cooling phase.

[0153] E6. A method for recovering a recombinant protein during a biomanufacturing process, comprising: establishing a cell culture by inoculating a bioreactor with mammalian cells expressing the recombinant protein; maintaining the cell culture during a growth phase and a production phase, wherein the cell culture is mixed with an exogenous protease inhibitor during the growth phase and / or during or after the production phase; mixing the cell culture with an acidic solution to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and Incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, The recombinant protein contains an asparaginyl endopeptidase (AEP) cleavage site. method.

[0154] E7. The method of E5 or E6, wherein the exogenous protease inhibitor is an AEP inhibitor.

[0155] E8. The method of any one of E1-E7, wherein the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and any combination of the foregoing.

[0156] E9. The method of any one of E1-E8, comprising incubating the acidified cell culture for at least about 60 minutes.

[0157] E10. The method according to any one of E1 to E9, comprising incubating the acidified cell culture for about 60 minutes to about 48 hours.

[0158] E11. The method of any one of E1-E10, further comprising separating one or more cell culture impurities from the acidified cell culture to obtain a clarified cell culture.

[0159] E12. The method of E11, wherein one or more cell culture impurities are separated from the acidified cell culture by continuous solids discharge centrifugation, disc stack centrifugation, and / or depth filtration.

[0160] E13. Deep filtration at approximately 150 L / m 2 Less than (e.g., about 125 L / m 2 The method according to E12, wherein the loading dose is less than 100 mg / kg.

[0161] E14. The method of any one of E1 to E13, wherein the one or more cell culture impurities are selected from host cell proteins and nucleic acids.

[0162] E15. The method according to any one of E1 to E14, wherein the pH of the acidified cell culture is from about 4.9 to about 5.2.

[0163] E16. The method according to any one of E1 to E15, wherein the pH of the acidified cell culture is about 5.0 to about 5.2.

[0164] E17. The method according to any one of E1 to E16, wherein the pH of the acidified cell culture is about 5.1.

[0165] E18. The method of any one of E1-E17, wherein the pH of the acidified cell culture deviates by less than about ±0.2 pH units during incubation.

[0166] E19. The method of any one of E1 to E18, wherein the acidified cell culture comprises a recombinant protein comprising a sequence upstream of the AEP cleavage site and a sequence downstream of the AEP cleavage site.

[0167] E20. The method of any one of E1-E19, wherein the acidified cell culture has a reduced amount of one or more fragments of the recombinant protein compared to an alternative acidified cell culture having a pH below about 4.6.

[0168] E21. The method of any one of E1-E20, wherein less than about 5 wt% (e.g., less than about 4 wt%; less than about 3 wt%; less than about 2 wt%) of the recombinant protein in the acidified cell culture is fragments of the recombinant protein, e.g., as detected by UV absorbance relative peak area in reduced capillary electrophoresis.

[0169] E22. The method of any one of E1-E19, wherein less than about 1 w / w% of the recombinant protein in the acidified cell culture is a fragment of the recombinant protein.

[0170] E23. The method of E21 or E22, wherein the fragment of the recombinant protein comprises a species cleaved at the AEP cleavage site.

[0171] E24. The method according to any one of E1 to E23, further comprising measuring the amount or activity of AEP before mixing.

[0172] E25. The method of any one of E1-E23, further comprising measuring the amount of AEP proenzyme prior to mixing.

[0173] E26. The method of claim E24 or E25, wherein the amount of AEP or AEP proenzyme is measured using mass spectrometry or an immunoassay.

[0174] E27. The method of E24, wherein the activity of AEP is measured using a cleavage assay.

[0175] E28. A method for inhibiting cleavage of a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site during a biomanufacturing process involving acid precipitation, comprising: Measuring the amount or activity of AEP in a sample isolated from a cell culture operation, a harvesting operation, or a purification operation of a biomanufacturing process; and If the amount or activity of AEP exceeds the threshold, adjust the pH of the acid precipitation to about 4.6 to about 5.3. A method comprising:

[0176] E29. The method of E28, wherein cleavage of the recombinant protein is inhibited relative to recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6.

[0177] E30. The method of E28 or E29, wherein the sample is isolated from a cell culture or harvesting operation.

[0178] E31. The method according to any one of E28 to E30, wherein the amount of AEP is measured using mass spectrometry or immunoassay.

[0179] E32. The method according to any one of E28 to E30, wherein the activity of AEP is measured using a cleavage assay.

[0180] E33. The method of any one of E28 to E32, further comprising adjusting the dissolved oxygen level of the cell culture to between about 64 mmHg and about 128 mmHg during the cell culture cooling operation of the biomanufacturing process if the amount or activity of AEP exceeds a threshold value.

[0181] E34. The method of any one of E28 to E33, further comprising admixing an exogenous protease inhibitor (e.g., an exogenous AEP inhibitor) with the cell culture during the growth and / or production phase of the biomanufacturing process if the amount or activity of AEP is above a threshold value.

[0182] E35. The method according to any one of E28 to E34, wherein the threshold amount of AEP is greater than about 1000 ng AEP per mg of harvest cell culture fluid (HCCF).

[0183] E36. The method according to any one of E28 to E35, wherein the threshold amount of AEP is greater than about 2000 ng of AEP per mg of harvested cell culture fluid (HCCF).

[0184] E37. The method according to any one of E28 to E36, wherein the threshold activity of AEP exceeds about 3% clipping of the recombinant protein at the AEP cleavage site.

[0185] E38. A method for inhibiting cleavage of a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site during a biomanufacturing process involving acid precipitation, comprising: Measuring the amount of one or more fragments of a recombinant protein in a sample isolated during a cell culture operation, a harvesting operation, or a purification operation of a biomanufacturing process; and If the amount of one or more fragments exceeds the threshold, adjust the pH of the acid precipitation to about 4.6 to about 5.3. A method comprising:

[0186] E39. The method of E38, wherein cleavage of the recombinant protein is inhibited relative to recombinant protein produced by an alternative biomanufacturing process using an acid precipitation pH of less than about 4.6.

[0187] E40. The method of E38 or E39, wherein the sample is isolated during a cell culture or harvesting procedure.

[0188] E41. The method of any one of E38 to E40, further comprising adjusting the dissolved oxygen level of the cell culture to between about 64 mmHg and about 128 mmHg during the cell culture cooling operation of the biomanufacturing process if the amount of one or more fragments exceeds a threshold value.

[0189] E42. The method of any one of E38 to E41, further comprising admixing an exogenous protease inhibitor (e.g., an exogenous AEP inhibitor) with the cell culture during the growth and / or production phase of the biomanufacturing process if the amount of one or more fragments is above a threshold value.

[0190] E43. The method of any one of E38 to E42, wherein the threshold is at least about 1 w / w% of the recombinant protein being one or more fragments.

[0191] E44. The method according to any one of E38 to E43, wherein the pH of the acid precipitation is from about 4.9 to about 5.2.

[0192] E45. The method according to any one of E38 to E44, wherein the pH of the acid precipitation is about 5.0 to about 5.2.

[0193] E46. The method according to any one of E38 to E45, wherein the pH of the acid precipitation is about 5.1.

[0194] E47. The method of any one of E1-E46, wherein the recombinant protein is an antigen-binding protein.

[0195] E48. The method according to any one of E1 to E47, wherein the recombinant protein is an antibody.

[0196] E49. The method according to any one of E1 to E48, wherein the recombinant protein is an IgG2 antibody.

[0197] E50. The method of any one of E1 to E49, wherein the recombinant protein comprises an IgG2 heavy chain constant region.

[0198] E51. The method of any one of E1 to E50, wherein the recombinant protein comprises a wild-type IgG2 heavy chain constant region.

[0199] E52. The method of any one of E1-E50, wherein the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0200] E53. The method of any one of E1 to E52, wherein the recombinant protein comprises the CH1 domain of an IgG2 heavy chain constant region.

[0201] E54. The method of any one of E1 to E53, wherein the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0202] E55. The method of any one of E1-E54, wherein the biomanufacturing process comprises a perfusion cell culture process.

[0203] E56. The method of any one of E1-E54, wherein the biomanufacturing process comprises an enriched fed-batch cell culture process.

[0204] E57. The method of any one of E1-E54, wherein the biomanufacturing process comprises an intensified cell culture process.

[0205] E58. The biomanufacturing process should be carried out at a viable cell density of at least approximately 10 6 The method according to any one of E1 to E57, comprising a production phase in which the culture medium is 100 cells / mL.

[0206] E59. The biomanufacturing process is carried out at a viable cell density of at least approximately 10 7 The method according to any one of E1 to E58, comprising a production phase in which the culture medium is 100 cells / mL.

[0207] E60. The method of any one of E1-E59, wherein the biomanufacturing process includes a production phase in which the compacted cell mass is between about 2% and about 40%.

[0208] E61. The method of any one of E1-E60, wherein the biomanufacturing process includes a production phase carried out in a bioreactor having a volume of at least about 500 L.

[0209] E62. The method of any one of E1-E61, wherein the biomanufacturing process includes a production step carried out in a bioreactor having a volume of at least about 2 kL. [Brief explanation of the drawings]

[0210] [Figure 1]Shown are AEP levels from in-process samples taken during pilot-scale production of an IgG2 antibody (mAb1), which process included an acid precipitation step at a pH of about 4.6. [Figure 2] Fragmentation levels as measured by LC-MS are shown. The intact peptides (LMSTN325DLK (SEQ ID NO: 1) and LD305LTPSPE311VPLTILK (SEQ ID NO: 2)) reflect the proenzyme form of AEP. After acid precipitation, the intensity of these peptides was reduced approximately 100-fold, indicating that AEP was cleaved at N325, D305, and E311 to its mature, active form. [Figure 3] mAb1 fragmentation as measured by LC-MS is shown. DETAILED DESCRIPTION OF THE INVENTION

[0211] Definition: In some embodiments, "about," when used in connection with a measurable, numerical variable, refers to the indicated value of the variable and all values ​​of the variable that are within experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or ±10% of the indicated value, whichever is greater. In some embodiments, numerical ranges are inclusive of the numbers (i.e., endpoints) that define the range.

[0212] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0213] As used herein, the terms "a" and "an" mean "one or more" unless specifically stated otherwise. In addition, "one or more" and "at least one" are used interchangeably herein. Further, unless the context requires otherwise, singular terms include the plural and plural terms include the singular.

[0214] As used herein, the term "acid precipitation" refers to a recovery operation in which the pH of a cell culture is lowered to induce precipitation of one or more cell culture impurities.

[0215] As used herein, "asparaginyl endopeptidase", also referred to as "AEP", "legumain", and "δ-secretase", is a lysosomal cysteine endopeptidase from the C13 peptidase family that hydrolyzes substrates at the C-terminus of asparagine residues at weakly acidic pH (2.5 < pH < 4.5). AEP is converted from an inactive proenzyme form to a mature active form by autoproteolysis of the C-terminal portion at weakly acidic pH. Zhao et al, Cell Research (2014) 24:344-358. The AEP of Chinese hamster ovary (CHO) cells is predicted to have the following sequence including a signal peptide.

Chemical formula

[0216] In Zhao et al., Cell Research (2014) 24:344-358, the AEP sequences of mouse (M. musculus), human (H. sapiens), and wild pig (S. scrofa) were aligned to create a universal numbering scheme for AEP enzymes. The sequence of CHO AEP is three residues longer due to three additional residues in the signal peptide at the N-terminus. Thus, CHO AEP residues N328, D308, and E314 in SEQ ID NO: 3 correspond to N325, D305, and E311 in the universal AEP numbering scheme proposed by Zhao et al., Cell Research (2014) 24:344-358 for AEPs from mouse, human, and wild pig.

[0217] As used herein, the term "antigen-binding protein" refers to a protein or polypeptide that comprises an antigen-binding region or portion that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include, but are not limited to, antibodies, fusion proteins, VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2, or "rIgG" (a "half antibody" consisting of a heavy and light chain), or modified antigen-binding portions of full-length antibodies, such as three-chain antibody-like molecules, heavy chain only antibodies, and the like. antibody), single-chain variable fragments (scFv), di-scFv or bi(s)scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, "minibodies" exemplified by the following structures: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multibodies, e.g., triabodies or tetrabodies, and single domain antibodies, e.g., nanobodies or single variable domain antibodies comprising only one variable region which may be VHH, VH, or VL, which specifically bind to an antigen or target independent of other variable regions or domains.

[0218] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa).

[0219] As used herein, the term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from the amino-terminus (N-terminus) to the carboxyl-terminus (C-terminus), a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain.

[0220] As used herein, the term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG class antibodies and IgA class antibodies are further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0221] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy-light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein. From the N-terminus to the C-terminus, both naturally occurring light-chain variable regions and heavy-chain variable regions typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the CDRs and FRs of a given antibody. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0222] Papain digestion of an antibody produces two identical antigen-binding proteins called "Fab" fragments (each of which has a single antigen-binding site) and the remaining "Fc" fragment (which contains all but the first domain of the immunoglobulin heavy chain constant region). The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH domain and CH1 domain from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0223] An "Fc fragment" or "Fc region" of an immunoglobulin generally comprises two constant domains (a CH2 domain and a CH3 domain), and optionally comprises a CH4 domain. The Fc region may be derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0224] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by inter-heavy chain disulfide bridges at the hinge region.

[0225] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy-chain variable region (VH) and one immunoglobulin light-chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light- or heavy-chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site comprising both the VH and VL.

[0226] A "single-chain variable antibody fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0227] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks and nurse sharks, and Camelidae, such as camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region; i.e., these functional antibodies are heavy chain homodimers with only the structure H2L2 (also referred to as "heavy chain antibodies" or "HCAbs"). Camelized VHHs have been reported to recombine with IgG2 and IgG3 constant regions containing hinge, CH2, and CH3 domains but lacking the CH1 domain. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold, providing a long transmembrane loop structure in the framework.

[0228] As used herein, the term "heavy chain-only antibody" refers to an immunoglobulin protein consisting of two heavy chain polypeptides (e.g., heavy chain polypeptides of approximately 50-70 kDa each). "Heavy chain-only antibodies" lack the two light chain polypeptides found in conventional antibodies. Heavy chain-only antibodies constitute approximately one-quarter of the IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These molecules are formed by two heavy chains and lack light chains. The resulting variable antigen-binding portion, referred to as a VHH domain, represents the smallest naturally occurring intact antigen-binding site and is only approximately 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Immunization can generate heavy-chain antibodies with high specificity and affinity against various antigens (van der Linden, RH, et al., Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al., J. Biotechnol. 78, 11-21 (2000)). The levels of expression, solubility, and stability of these antibodies are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA, et al., FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in these antibodies, called VNAR (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0229] In some embodiments, a "heavy chain-only antibody" is a dimeric antibody comprising a VH antigen-binding domain and CH2 and CH3 constant domains, with the CH1 domain absent. In some embodiments, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. The heavy chain-only antibodies described herein may belong to the IgG subclass, although heavy chain-only antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In some embodiments, the heavy chain-only antibody may be of the IgG1, IgG2, IgG3, or IgG4 subtype, for example, the IgG1 or IgG4 subtype. In some embodiments, the heavy chain-only antibody is of the IgG1 or IgG4 subtype, and one or more of the CH domains have been modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG4 subtype, and one or more of the CH domains have been modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG1 subtype, and one or more of the CH domains have been modified to alter the effector function of the antibody. Modifications of CH domains to alter effector function are further described herein. Non-limiting examples of heavy chain-only antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0230] As used herein, the term "tri-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or an antigen-binding fragment of such antibody chains, each of which comprises an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion, absent a CH1 domain, comprising CH2, and / or CH3, and / or CH4 domains, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domains comprise, consist essentially of, or consist of a heavy-chain-only antibody derived from or sharing sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions include V H and / or V L Gene segments, D and J H Gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0231] As used herein, the term "bioreactor" refers to any vessel useful for growing cell cultures (e.g., mammalian or bacterial cell cultures). "Bioreactor" as used herein encompasses the term "fermentor" (i.e., a vessel useful for growing bacterial cell cultures that typically includes a more powerful agitator and increased gas flow compared to vessels used for growing mammalian cell cultures). Non-limiting examples of bioreactors include stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. In some embodiments, an exemplary bioreactor may perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients and / or a carbon source, injecting an appropriate gas (e.g., oxygen, etc.), inflow and outflow of fermentation or cell culture medium (e.g., perfusion of fresh cell culture medium and removal of spent cell culture medium), separation of gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Unless the context dictates otherwise, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable bioreactor diameter may be used. Unless the context dictates otherwise, in some embodiments, the bioreactor may have a volume between 100 mL and 50,000 L. Unless the context dictates otherwise, a bioreactor can be of any size as long as it is useful for culturing cells; typically, the bioreactor is sized appropriately for the volume of cell culture to be grown therein. In a non-limiting embodiment, and unless the context dictates otherwise, the bioreactor can be at least 1 liter (L), or can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000, 20,000, or more, or any volume therebetween. The internal conditions of the bioreactor (e.g., but not limited to, pH, dissolved oxygen (DO), and temperature) can be controlled during the culturing period.Those skilled in the art will be able to recognize and select a suitable bioreactor for use in the methods disclosed herein based on relevant considerations.

[0232] As used herein, the term "cell culture" or "culturing" refers to the growth and proliferation of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian and bacterial cells are known in the art (see, e.g., Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992)). Mammalian cells may be cultured in suspension or attached to a solid culture medium. In some embodiments, fluidized-bed bioreactors, hollow-fiber bioreactors, roller bottles, shake flasks, and / or stirred-tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0233] As used herein, the term "cell culture medium" (also referred to as "culture medium," "culture medium," "cell culture medium," "tissue culture medium," etc.) refers to any nutrient solution used to grow cells (e.g., bacterial cells or mammalian cells). Cell culture media generally provide one or more of the following components: an energy source (e.g., in the form of carbohydrates, such as glucose); one or more essential amino acids (e.g., all essential amino acids, the 20 basic amino acids plus cysteine); vitamins and / or other organic compounds, which are typically required at low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, which are typically required at very low concentrations, e.g., concentrations in the micromolar range. As used herein, cell culture medium encompasses nutrient solutions typically used in and / or known for use with any cell culture process (e.g., without limitation, batch, extended batch, fed-batch, enriched, and / or perfusion, or continuous culture of cells).

[0234] As used herein, the term "cell density" refers to the number of cells in a given volume of culture medium. "Viable cell density" refers to the number of viable cells in a given volume of culture medium as determined by a standard viability assay (e.g., trypan blue exclusion) and can be measured at any time during a particular stage of the cell culture process. As used herein, the term "packed cell volume" (PCV), also referred to as "packed cell volume percentage" (PCV%), refers to the ratio of the volume of cells to the total volume of the cell culture, expressed as a percentage (Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter, and an increase in packed cell volume can occur by increasing cell density, cell diameter, or both. Packed cell volume is an indicator of the solid content in a cell culture. Because host cells vary in size and cell cultures also contain dead and dying cells and other cellular debris, compressed cell volume may more accurately describe the solids content within a cell culture.

[0235] As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a particular host cell (e.g., a mammalian host cell). Vectors can include viral vectors, non-episomal mammalian vectors, plasmids, and other non-viral vectors. Expression vectors can contain sequences that affect or control transcription, translation, and, if present, RNA splicing of an operably linked coding region. "Operably linked" means that the components to which this term is applied are in a relationship allowing them to perform their inherent functions. For example, a control sequence (e.g., a promoter) in a vector "operably linked" to a protein-coding sequence is positioned so that normal activity of the control sequence results in transcription of the protein-coding sequence and recombinant expression of the encoded protein.

[0236] As used herein, "fed-batch culture" refers to a form of suspension culture, specifically a method of culturing cells in which additional components are provided to the culture medium at some point or points after the initiation of the culture process. The provided components typically include nutritional supplements for the cells that are depleted during the culture process. Additionally or alternatively, the additional components may include supplemental components (e.g., cell cycle inhibitor compounds, etc.). In some embodiments, a fed-batch cell culture medium formulation contains components essential for cell survival and growth and may be enriched or concentrated relative to a basal cell culture medium formulation typically used to initiate the cell culture. The fed-batch culture may be stopped at some point, and the cells and / or components in the medium may be harvested and optionally purified.

[0237] As used herein, a "fusion protein" is a protein containing at least one polypeptide fused or linked to a heterologous polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is added in frame with a nucleotide sequence encoding a polypeptide sequence from a different protein, optionally separated from the sequence by a linker. The fusion gene can then be expressed by a recombinant host cell to produce the fusion protein. A fusion protein can include a fragment from an immunoglobulin protein (e.g., an Fc region fused or linked to a ligand polypeptide, receptor polypeptide, hormone, cytokine, growth factor, enzyme, or other polypeptide that is not a component of an immunoglobulin).

[0238] As used herein, the "growth phase" of a cell culture refers to the period of exponential cell growth (i.e., log phase) when cells are generally dividing rapidly.

[0239] As used herein, the term "harvested cell culture fluid" refers to a solution that has been treated by one or more operations to separate cells, cell debris, or other large particulates from recombinant protein. Such operations include, but are not limited to, chilling, flocculation, acidification, centrifugation, neutralization, sonication, and various forms of filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration), as described above. Harvested cell culture fluid includes cell culture lysate and cell culture supernatant. Harvested cell culture fluid may be further clarified to remove small particulate matter and soluble aggregates by filtration through a membrane having a pore size of about 0.1 μm to about 0.5 μm (e.g., a membrane having a pore size of about 0.22 μm).

[0240] As used herein, a "host cell" refers to a cell that has been transformed or can be transformed with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of a parent cell, regardless of whether the morphology or genetic make-up of the progeny is identical to that of the original parent cell, so long as the gene of interest is present. For example, a host cell that contains a nucleic acid encoding a recombinant protein operably linked to at least one expression control sequence (e.g., a promoter or enhancer) is a "recombinant host cell." When cultured under appropriate conditions, the host cell can synthesize the recombinant protein, which can then be collected from the culture medium (if the host cell secretes it into the medium) or directly from the producing host cell (if it is not secreted).

[0241] As used herein, a "low molecular weight" or "LMW" species of a recombinant protein of interest refers to fragments, truncated forms, or other incomplete variants of the recombinant protein that have a reduced molecular weight relative to the molecular weight of the intact, fully assembled form of the recombinant protein. LMW species can include, but are not limited to, proteolytic fragments, truncated forms resulting from cellular expression of mRNA splice variants, and single component polypeptides in the case of multi-chain polypeptide proteins (e.g., species of only the light or heavy chain when the recombinant protein is an antibody).

[0242] As used herein, "perfusion" cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion or continuous culture methods and that is sufficiently complete to support the cell culture during this process. In some embodiments, perfusion cell culture medium formulations may be enriched or concentrated relative to basal cell culture medium formulations to accommodate methods used to remove spent medium. In some embodiments, perfusion cell culture medium may be used in both the growth and production phases.

[0243] As used herein, the term "polypeptide" refers to a polymer of amino acids comprising at least 50 amino acids (eg, at least 100 amino acids).

[0244] As used herein, "production" cell culture medium refers to a cell culture medium typically used in a cell culture during the transition when exponential growth ends and protein production becomes dominant (i.e., the "transition" and / or "production" phase), and that is sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase. The production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of the cell culture.

[0245] As used herein, the "production phase" of a cell culture refers to the period after logarithmic cell growth has ended and production of recombinant protein predominates.

[0246] As used herein, the term "protease inhibitor" refers to a molecule that at least partially inhibits the function of one or more protein-based enzymes that cleave other proteins. In some embodiments of the present disclosure, an exogenous protease inhibitor is added to a cell culture to inhibit cleavage of a recombinant protein of interest. In some embodiments, the exogenous protease inhibitor is a commercially available protease inhibitor cocktail intended to increase the stability of secreted proteins, such as E-64 protease inhibitor, TCM ProteaseArrest™ Protease Inhibitor Cocktail (G-Biosciences), Protease Inhibitor Cocktail I (R&D Systems), Halt™ Protease Inhibitor Cocktail (Thermo Scientific), or Protease Inhibitor Cocktail (Promega or Sigma-Aldrich). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor. In some embodiments, the AEP inhibitor is AENK, which is commercially available, for example, from Sigma-Aldrich. In some embodiments, the AEP inhibitor is δ-secretase inhibitor 11 (7-morpholin-4-yl-benzo[1,2,5]oxadiazol-4-ylamine). In some embodiments, the AEP inhibitor is 7-morpholinobenzo[c][1,2,5]oxadiazol-4-amine. In some embodiments, the AEP inhibitor is a substituted 3,7-dihydropurine-2,6-dione derivative described in U.S. Patent Application Publication No. 2017 / 0166569.

[0247] As used herein, the term "recombinant protein" refers to a heterologous protein produced by a host cell transfected with a nucleic acid encoding the protein when the host cell is grown in cell culture.

[0248] As used herein, the term "unit operation" refers to a functional step performed as part of the process of purifying a recombinant protein of interest. A unit operation can be designed to accomplish a single purpose or multiple purposes, such as capture and viral inactivation steps. A unit operation can also include holding or storage steps between processing steps.

[0249] host cell The cell lines (also referred to as "cells" or "host cells") used in this disclosure are genetically engineered to express recombinant proteins for commercial or scientific purposes. The cells may be suitable for adherent, monolayer, and / or suspension culture, transfection, and expression of recombinant proteins, such as antibodies. The cells may be used, for example, with batch, fed-batch, and perfusion or continuous culture methods. Such cells are typically cell lines obtained or derived from mammals and are capable of growing and surviving when placed in either monolayer or suspension culture in media containing appropriate nutrients and / or other factors (e.g., those described herein). Typically, cells are selected that can express and secrete proteins or that can be molecularly engineered to express and secrete large amounts of a particular protein (more specifically, a glycoprotein of interest) into the culture medium. The selection of an appropriate host cell for expression of a recombinant protein depends on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) desired or essential for activity, and the ease of folding into a biologically active molecule. In some embodiments of the disclosed methods, the host cell is a mammalian host cell.

[0250] Cell lines are typically derived from lineages arising from primary cultures that can be maintained in culture for an unlimited period of time. Cells may contain an expression vector (construct), such as a plasmid, introduced by, for example, transformation, transfection, infection, or injection, which carries a coding sequence or a portion thereof encoding a protein for expression and production during the culture process. Such expression vectors contain elements necessary for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding the proteins and polypeptides to be produced and appropriate transcription and translation control elements can be constructed using methods well known and practiced by those skilled in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4 th edition Cold Spring Harbor Press, Plainview, NY or any earlier edition; F.M.A. Masubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY or any earlier edition; Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for all purposes.

[0251] Suitable host cells include, but are not limited to, those commercially available from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0252] In some embodiments, the host cell is selected from CHO cells. CHO cells, such as CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems enable high-level recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Also included is the glutamine synthase (GS) knockout CHOK1SV cell line, which utilizes glutamine synthase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells for use in the biomanufacturing processes disclosed herein include, but are not limited to, the following (ECACC accession numbers in parentheses): CHO (85050302), CHO (PROTEIN FREE) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhFr- (94060607), CHO / dhFr-AC-free (05011002), and RR-CHOKI (92052129).

[0253] To generate a host cell line (e.g., a mammalian cell line) engineered to express a recombinant protein of interest, one or more nucleic acids encoding the recombinant protein (or its components, in the case of a multi-chain protein) are first inserted into one or more expression vectors. Nucleic acid control sequences useful in expression vectors for expression in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence may also optionally be encoded by the expression vector and operably linked to the coding sequence of interest, thereby enabling the recombinant host cell to secrete the expressed protein, so that the recombinant protein can be more easily isolated from the cells, if desired. A vector may also contain one or more selectable marker genes to facilitate selection of host cells into which the vector has been introduced. In some embodiments, vectors employing a protein fragment complementation assay using a protein reporter, such as dihydrofolate reductase, are used (see, e.g., U.S. Pat. No. 6,270,964). Suitable mammalian expression vectors are known in the art and are commercially available.

[0254] Typically, vectors used in any of the host cells contain sequences for maintaining the plasmid and for cloning and expressing exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a native or heterologous signal peptide sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. Vectors can be constructed from a starting vector, such as a commercially available vector, or additional elements can be obtained separately and ligated into the vector.

[0255] Culture method A variety of culture methods can be used to produce the recombinant protein of interest, including, but not limited to, batch culture, fed-batch culture, perfusion culture, and enriched cell culture.

[0256] Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time, followed by a total harvest. Cultures grown using batch methods undergo an increase in cell density until a maximum cell density is reached, after which the viable cell density declines as medium components are consumed and levels of metabolic by-products (e.g., lactate and ammonia) accumulate. Harvesting is typically initiated at a maximum cell density (e.g., 5×10, depending on medium composition, cell line, etc.). 6 The batch process is the simplest culture method, but viable cell density is limited by nutrient availability, and once the cells reach maximum density, the culture declines and production decreases. In batch culture, the production phase cannot be extended, typically about 3 to 7 days, because waste accumulation and nutrient depletion lead to rapid culture decline.

[0257] Fed-batch culture improves on the batch process by using a bolus or continuous medium feed to replenish consumed medium components. Because fed-batch culture receives additional nutrients throughout the run, it can achieve higher cell densities (10-30 x 10 depending on medium composition, cell line, etc.) compared to batch methods. 6Fed-batch cultures have the potential to achieve greater than 1000 cells / mL (>1000 cells / mL) and increased product titers. Unlike batch processes, by manipulating the feeding strategy and medium composition, a two-phase culture can be created and maintained, distinguishing between a cell growth phase (growth phase) to achieve a desired cell density and a period during which cell growth is halted or slow (production phase). Therefore, fed-batch cultures have the potential to achieve higher product titers compared to batch cultures. Typically, batch methods are used during the growth phase and fed-batch methods during the production phase, but fed-batch feeding strategies can be used throughout the process. However, unlike batch processes, the volume of the bioreactor is a limiting factor, restricting the amount of feed. Also, like batch processes, accumulation of metabolic by-products can lead to culture decline, which often limits the duration of the production phase to approximately 10–21 days. Fed-batch cultures are discontinuous, and harvesting typically occurs when metabolic by-product levels or culture viability reach a predetermined level. Compared to unfed batch cultures, fed-batch cultures can produce higher amounts of recombinant protein (see, eg, US Pat. No. 5,672,502).

[0258] Perfusion methods offer a potential improvement over batch and fed-batch methods by adding fresh medium and simultaneously removing spent medium during cultivation. A typical perfusion culture begins with a batch culture start-up lasting 1-2 days, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture with simultaneous removal of spent medium, retaining cells and additional high-molecular-weight compounds, such as proteins (based on the filter's molecular weight cutoff), throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent medium while maintaining cell density. Non-limiting examples of filtration methods include alternating tangential flow filtration and recirculating tangential flow. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, e.g., U.S. Pat. No. 6,544,424; Furey, 2002, Gen. Eng. News. 22(7):62-63.

[0259] Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate can be less than to many working volumes per day. The cells are retained in culture, and the removed spent medium is substantially cell-free or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture.

[0260] In a typical large-scale commercial cell culture strategy, approximately one-third to over one-half of the reactor volume is filled with biomass, 30–90(+) × 10 6 The goal is to achieve high cell densities, such as 1 × 10 cells / mL. 8 Very high cell densities, exceeding 100 cells / mL, have been achieved. A potential advantage of perfusion processes is the ability to maintain production cultures for longer periods of time compared to batch or fed-batch culture methods. However, maintaining long-term perfusion cultures requires increased medium preparation, use, storage, and disposal, especially for cultures at high cell densities that also require more nutrients. All of this can increase production costs compared to batch and fed-batch methods. Furthermore, high cell densities can cause issues during production, such as maintaining dissolved oxygen levels, as well as problems with increased aeration, including more oxygen supply and more carbon dioxide removal, which can result in more foaming and the need for changes in anti-foaming strategies, and problems in harvesting and downstream processes, where the work required to remove excess cellular material can result in product loss, potentially negating the benefit of increased titer from increased cell mass.

[0261] Suitable culture conditions for mammalian cells, such as temperature, dissolved oxygen content, agitation rate, etc., are known in the art and may vary depending on the phase or stage of the cell culture. In some embodiments, the methods disclosed herein further include taking samples during the cell culture process and evaluating the samples to quantitatively and / or qualitatively monitor characteristics of the recombinant protein and / or the cell culture process. In some embodiments, the samples are monitored quantitatively and / or qualitatively using process analytical techniques. For example, dissolved oxygen levels may be monitored during the cell culture process using methods known in the art, such as, for example, elementary chemical analysis (titration), electrochemical analysis (diaphragm electrode), and photochemical analysis (fluorescence).

[0262] During recombinant protein production, it is desirable to have a controlled system for growing cells for a desired period of time or to a desired density and then switching the physiological state of the cells to a growth-limited or arrested high-production state, in which the cells use energy and growth medium while increasing cell density to produce the recombinant protein. For commercial-scale cell culture and biotherapeutic manufacturing, the ability to limit or arrest cell growth and maintain cells in a growth-limited or arrested state during the production phase is highly desirable. Such methods include, for example, temperature shift, use of chemical inducers of protein production, nutrient limitation, or starvation and cell cycle inhibitors, either alone or in combination. For example, a typical cell culture undergoes a growth phase, which is an exponential growth phase during which cell density increases. During the growth phase, cells are cultured in cell culture medium containing necessary nutrients and additives under conditions that achieve optimal growth for the particular cell line (typically a temperature of about 25-40°C in a humidified, controlled atmosphere). Cells are typically maintained in the growth phase for 1 to 8 days, e.g., 3 to 7 days, e.g., 7 days. The length of the growth phase for a particular cell line can be determined by one of skill in the art and is generally a period sufficient for the particular cells to propagate to a viable cell density within the range of about 20% to 80% of the maximum viable cell density possible when the culture is maintained under growth conditions. The growth phase is followed by a transition phase, during which exponential cell growth slows and protein production begins to increase. This marks the beginning of the stationary phase (production phase), during which cell density typically levels off and product titer increases. During the production phase, the medium is typically replenished to support continued production of the recombinant protein.

[0263] In certain embodiments of the disclosed methods, culture conditions may be adjusted to facilitate the transition of a cell culture from the growth phase to the production phase. For example, the growth phase of a cell culture may occur at a higher temperature than the production phase of the cell culture. In some embodiments, the growth phase may occur at a first temperature of about 35°C to about 38°C, and the production phase may occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In one embodiment, a temperature shift from about 35°C to about 37°C to a temperature of about 31°C to about 33°C may be used to facilitate the transition of a culture from the growth phase to the production phase. For example, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with, before, and / or after the temperature shift, or may be added in place of the temperature shift. If the inducer is added after the temperature shift, the inducer may be added between 1 hour and 5 days after the temperature shift, optionally between 1 and 2 days after the temperature shift.

[0264] Additionally, any cell culture medium capable of supporting the growth of suitable host cells in culture may be used. Typically, cell culture media contain buffers, salts, an energy source, amino acids, vitamins, and essential trace elements. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells, are commercially available and include, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media (e.g., EX-CELL™ 300 series), which may be obtained from the American Type Culture Collection or SAFC Biosciences and other vendors. Cell culture media may be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture medium can also be enriched by adding nutrients or other supplements, which can be used at concentrations higher than the usual recommended concentrations. In certain embodiments, the culture medium used in the method of the present disclosure is a chemically defined medium, which refers to a cell culture medium in which all components have known chemical structures and concentrations. Chemically defined media are typically serum-free and do not contain hydrolysates or animal-derived components.

[0265] Various media formulations may be used during the culture period, for example, to facilitate the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase) and / or to optimize conditions during cell culture (e.g., concentrated media provided during perfusion culture). Growth media formulations may be used to promote cell growth and minimize protein expression. Production media formulations may be used to promote production of the recombinant protein of interest and cell maintenance while minimizing the growth of new cells. A feed medium is a cell culture medium that typically contains more concentrated components (e.g., nutrients and amino acids) that are consumed during the production phase of the cell culture. A feed medium may be used to replenish and maintain an active culture, particularly a culture operated in fed-batch, semi-perfusion, or perfusion mode. Such concentrated feed medium may contain most of the components of the cell culture medium, for example, about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x their normal amounts.

[0266] In some embodiments of the disclosed methods, the mammalian cells are cultured for a period of time during which the recombinant protein is expressed and secreted by the mammalian cells. This period (i.e., the duration of the production phase of the cell culture) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the production phase of the cell culture is about 7 to about 28 days, about 10 to about 30 days, about 7 to about 14 days, about 10 to about 18 days, about 3 to about 15 days, about 5 to about 8 days, about 12 to about 15 days, about 12 to about 18 days, or about 15 to about 21 days. In some embodiments, the duration of the production phase of the cell culture is 7, 8, 9, 12, 15, 18, or 21 days.

[0267] In some embodiments of the disclosed methods, the biomanufacturing process produces cells with a viable cell density of at least 100×10 5 cells / mL, e.g., approximately 100 x 10 5cells / mL ~ approx. 10 x 10 7 cells / mL, approximately 250 x 10 5 cells / mL ~ approx. 900 x 10 5 cells / mL, approximately 300 x 10 5 cells / mL ~ 800 x 10 5 cells / mL, or approximately 450 x 10 5 cells / mL ~ 650 x 10 5 The viable cell density is determined by staining a culture sample with trypan blue, which is taken up only by dead cells. The total number of cells is then counted, and the viable cell density is determined by dividing the number of stained cells by the total number of cells and taking the reciprocal.

[0268] In some embodiments of the disclosed methods, the biomanufacturing process includes a production phase in which the compacted cell volume is 35% or less, hi some embodiments, the compacted cell volume is 30% or less.

[0269] Refining Process The expressed recombinant protein may be secreted into the culture medium from which it may be recovered and / or collected. Recovery procedures involving acid precipitation may be combined with additional recovery strategies, such as centrifugation, e.g., disk stack centrifugation, intermittent discharge centrifugation, or continuous solids discharge centrifugation; filtration, e.g., tangential flow filtration, microfiltration, ultrafiltration, and depth filtration; precipitation / sedimentation methods, e.g., flocculation; and chromatographic media-based separation.

[0270] In addition to recovery procedures involving acid precipitation, the present disclosure includes methods involving all known post-recovery techniques, including, for example, Protein A purification of immunoglobulins and immunoglobulin-like biologics, and chromatography-based separation and polishing steps including columns and alternative modes of chromatographic separation by ion exchange chromatography (IEX), e.g., anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, size exclusion chromatography (SEC), gel filtration, or any other known form of chromatographic separation of biological and / or biochemical substances.

[0271] In some embodiments of the disclosed methods, the recombinant protein recovered from the host cells or cell culture medium may be further purified or partially purified by one or more unit operations to remove cell culture medium components, host cell proteins or nucleic acids, or other process- or product-related impurities. One skilled in the art may select appropriate unit operations for further purifying the recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of the host cells in which the recombinant protein is expressed, and the composition of the culture medium in which the host cells are grown. By way of example, in some embodiments, the recombinant protein is purified from the recovered permeate by one or more of aggregation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0272] The capture unit operation may include capture chromatography, which utilizes resins and / or membranes containing agents that bind to the recombinant protein of interest, and may include, for example, affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), etc. Such chromatography materials are known in the art and commercially available. For example, if the recombinant protein is an antibody or contains an antibody-derived component (e.g., an Fc domain), affinity chromatography using ligands such as Protein A, Protein G, Protein A / G, or Protein L may be used as a capture chromatography unit operation to further purify the recombinant protein. In other embodiments, the recombinant protein of interest may contain a polyhistidine tag at its amino or carboxyl terminus and subsequently purified using IMAC. Recombinant proteins may be engineered to contain other purification tags, such as a FLAG® tag or a c-myc epitope, and then purified by affinity chromatography using specific antibodies directed against such tags or epitopes.

[0273] Additional unit operations to inactivate, reduce, and / or eliminate viral contaminants may include filtration processes and / or adjustments to solution conditions. One method for achieving viral inactivation is incubation at low pH (e.g., pH <4). A viral inactivation operation at low pH may be followed by a neutralization unit operation to readjust the virally inactivated solution to a pH more compatible with the requirements of subsequent unit operations. A viral inactivation operation at low pH may also be followed by filtration, such as depth filtration, to remove any turbidity or precipitate that may result. Adjustments to temperature or chemical composition (e.g., the use of surfactants) may also be used to achieve viral inactivation. Virus filtration may be performed using microfiltration or nanofiltration membranes, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0274] Polishing unit operations may utilize a variety of chromatographic methods for purifying the protein of interest and removing contaminants and impurities. Polish chromatography unit operations may utilize agent-containing resins and / or membranes that can be used in either "flow-through mode" (where the protein of interest is contained in the eluent and contaminants and impurities bind to the chromatographic medium) or "bind and elute mode" (where the protein of interest binds to the chromatographic medium and is eluted after the contaminants and impurities pass through or are washed off the chromatographic medium). Examples of such polish chromatography methods include, but are not limited to, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and size-exclusion chromatography (e.g., gel filtration).

[0275] The purified recombinant protein can be formulated, i.e., buffer exchanged, sterilized, bulk packaged, and / or packaged for the end user. For example, concentration of the acid and buffer exchange of the recombinant protein of interest into a desired formulation buffer for bulk storage of a drug substance or drug product can be achieved by ultrafiltration and diafiltration. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.

[0276] Recombinant proteins Any type of recombinant protein containing an AEP cleavage site can be recovered according to the methods of the present disclosure, including proteins containing a single polypeptide chain or multiple polypeptide chains. Recombinant proteins of the present disclosure include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. By way of example, recombinant proteins may include, but are not limited to, cytokines, growth factors, hormones, mutant proteins, fusion proteins, antibodies, antibody fragments, peptibodies, T-cell engaging molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0277] In other embodiments, the recombinant protein recovered according to the methods of the present disclosure is an antigen-binding protein, including, but not limited to, antibodies, peptibodies, antibody derivatives, antibody analogs, fusion proteins (e.g., single-chain variable fragments (scFvs), two-chain (bivalent) scFvs, and IgG scFvs (see, e.g., Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), hetero-IgGs (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Additional antigen binding proteins include, but are not limited to, bispecific T cell engager (BiTE®) molecules, bispecific T cell engagers with extensions such as half-life extensions (e.g., HLE BiTE molecules, HeteroIg BITE molecules), chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).

[0278] In some embodiments, the antigen binding protein binds to one or more of the following, alone or in any combination: CD proteins, including but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174, HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptor, EGFRvIII, cell adhesion molecules, such as LFA-1, M ol, p150,95, VLA-4, ICAM-1, VCAM, as well as alpha v / beta 3 integrin, growth factors, such as, but not limited to, vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Mullerian inhibitory factor, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factor, such as aF GF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), such as TGF-α and TGF-β, e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, among others, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors, insulin and insulin-related proteins, such as, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factors. Long factor binding proteins (coagulation and coagulation-related proteins, e.g., Factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, e.g., urokinase and tissue plasminogen activator (“t-PA”), bombadin, thrombin, thrombopoietin, and thrombopoietin receptors, colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF, among others, other blood and serum proteins, e.g., albumin, IgE,and blood group antigens, receptors, and receptor-associated proteins, such as flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor; neurotrophic factors, such as, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin, interferons, such as interferon-alpha, -beta, and -gamma, interleukins (IL), such as IL-1 through IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor, and / or IL-13 to receptor IL-13RA2, or IL-17 receptor, IL-1RAP; viral antigens, such as, but not limited to, AIDS envelope virus antigen, lipoprotein, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and beta, enkephalinase, BCMA, Ig kappa, ROR-1, ErBB2, mesothelin, RANTES (regulated upon activation and normally expressed and secreted by T cells), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin, and activin, integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay-accelerating factors (DAFs), AIDS envelopes, transport proteins, homing receptors, MICs (MIC-a, MIC-B), ULBP1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, antigen-binding proteins, Somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-Met, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand,PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (Ly), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIB / IIIb (PAC-1), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), celerosteine, and biologically active fragments or variants of any of the foregoing.

[0279] In other embodiments, the recombinant protein recovered according to the methods of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody.

[0280] In some embodiments, the antibody is selected from abrilumab, brazicumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplasmab, nemolizumab, ontamalimab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, taforecimab, tanezumab, tezepelumab, tremelimumab, utomilumab, and borazidomab. In some embodiments, the antibody is selected from denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody is erenumab. In some embodiments, the antibody is evolocumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is tezepelumab.

[0281] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0282] In some embodiments, the recombinant protein recovered according to the methods of the present disclosure comprises an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a wild-type IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises an engineered IgG2 heavy chain constant region.

[0283] In some embodiments, the recombinant protein comprises the CH1 domain of an IgG2 heavy chain constant region. In some embodiments, the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

[0284] Monitoring the amount or activity of AEP or LMW species Some embodiments of certain methods of the present disclosure include monitoring the amount or activity of AEP of a desired recombinant protein and / or monitoring the amount of LMW species.

[0285] In some embodiments, the amount of AEP is measured using mass spectrometry or immunoassay. In some embodiments, the amount of AEP is measured using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the amount of AEP is measured using nanoscale liquid chromatography-mass spectrometry (nanoLC-MS).

[0286] In some embodiments, the activity of AEP is measured using a cleavage assay. In some embodiments where the biomanufacturing process utilizes CHO cells, the activated self-cleavage site N 325 , D 305 , and E 311 (LMSTN 325 DLK (SEQ ID NO: 1), and LD 305 LTPSPE 311AEP peptides containing VPLTILK (SEQ ID NO: 2) can be used as indicators of cleavage and activation. AEP peptides containing alternative self-cleavage sites can be used to monitor the activation of biomanufacturing processes that utilize alternative mammalian host cells.

[0287] In some embodiments, the amount of AEP proenzyme is measured using mass spectrometry or immunoassay. In some embodiments, the amount of AEP proenzyme is measured using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the amount of AEP proenzyme is measured using nanoscale liquid chromatography-mass spectrometry (nanoLC-MS). In some embodiments, where the biomanufacturing process utilizes CHO cells, the activated autocleavage site N 325 , D 305 , and E 311 (LMSTN 325 DLK, and LD 305 LTSPE 311 VPLTILK) is used as an indicator of the cleavage and activation of the AEP proenzyme to mature AEP.

[0288] In some embodiments, the amount of LMW species is measured using mass spectrometry or immunoassays (e.g., bioassays and / or titer assays). In some embodiments, the amount of LMW species is measured using reduced capillary electrophoresis-sodium dodecyl sulfate methods. In some embodiments, the amount of LMW species is measured using reduced reversed-phase (RP) chromatography methods. In some embodiments, the amount of LMW species is measured using ultra-high pressure liquid chromatography methods. [Example]

[0289] In order that this disclosure may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the disclosure in any manner.

[0290] Example 1. AEP-induced fragmentation of an IgG2 antibody During the development of a high-yield biomanufacturing process in CHO cells for an IgG2 antibody (mAb1), relatively high levels of fragmentation were detected by rCE-SDS. Applicants determined that fragmentation occurred at the N192 / F193 site (EU numbering) of mAb1, which is a conserved motif in the CH1 domain of the IgG2 heavy chain. The human IgG2 CH1 domain is provided below as SEQ ID NO: 4, with relevant residues underlined. [ka]

[0291] By LC-MS proteomics, the presence of the enzyme asparaginyl endopeptidase (AEP), also known as legumain, in the biomanufacturing process pool was identified. AEP is a lysosomal cysteine endopeptidase, which exhibits fragmentation activity at a weak acidic pH (2.5 < pH < 4.5), and its activity at pH 5 or above is negligibly small (Zhao, L. et al., Cell Res 2014, 24(3), 344 - 358). AEP originally exists in a proenzyme form during cell culture, which is converted to the mature active form by autoproteolysis of the terminal domain at low pH. From the tests described below, the presence of AEP in the high-yield biomanufacturing process was confirmed, and it was revealed that AEP mainly cleaves mAb1 at low pH during the acid precipitation operation of the antibody recovery from the bioreactor. Acid precipitation is a common step used to precipitate cells, DNA, and other cell debris before downstream purification while maintaining the antibody in a soluble form. From LC-MS analysis, it was also found that the proenzyme form of AEP is converted to the mature active form by autoproteolysis of the terminal part at low pH during acid precipitation. Since the AEP enzyme activity is higher at low pH, an increase in the AP target pH from 4.6 to 5.1 resulted in a reduction in fragmentation, as demonstrated by the decrease in low molecular weight (LMW, measured by rCE-SDS assay) in the drug substance from approximately 5% to approximately 0.3%. In summary, this study identified AEP cleavage as the cause of fragmentation during the low pH acid precipitation operation of recombinant proteins containing AEP cleavage sites such as IgG2 antibodies, and provided an understanding based on the mechanism and mitigation strategies. In particular, the effect of AEP on IgG2 antibodies has not been reported so far, which is probably due to either the relatively low level of AEP produced by a lower yield process or the absence of the acid precipitation operation during cell culture harvest.

[0292] Method In-process samples of mAb1 and final drug substance (DS) samples were collected at various stages from upstream to downstream processing for three pilot-scale (2 kL bioreactor) lots: PSL1, PSL2, and PSL3. A nanoLC-MS-based impurity biology approach was used to identify and quantify AEP levels relative to the mAb1 product for all samples. Both mature and proenzyme forms of AEP were measured and correlated with mAb1 fragmentation levels at various stages of the process. For further confirmation, a mAb1 reference standard (RS) produced by a lower-yield biomanufacturing process was spiked with commercially available AEP, and the site and level of fragmentation was assessed and correlated with the amount of AEP spiked.

[0293] Sample preparation 500 μg of each sample was taken and placed in a 10K MWCO Amicon centrifugal filter tube. The yeast standard proteins glucose-6-phosphate dehydrogenase (G6PD) and inorganic pyrophosphatase (IPYR) were added to each sample at 200 and 100 ng / mg, respectively. All samples were spun at 14,000 rpm for 30 minutes using a Beckman Coulter microcentrifuge 18 centrifuge. 400 μL of denaturing buffer (6 M guanidine-HCl / pH 7.5 / 2 mM EDTA / 20 mM methionine / 250 mM Tris) and 6 μL of 0.5 M 1,4-dithiothreitol (DTT) were added to the samples, which were then incubated at 37°C for 30 minutes. The samples were then alkylated in the dark by adding 14 μL of 0.5 M iodoacetic acid (IAA) for 30 minutes. The alkylation was quenched by adding 8 μL of 0.5 M DTT. After quenching, the denaturation buffer was passed through a 10K MWCO Amicon centrifugal filter, spun at 14,000 rpm for 30 minutes. The flow-through was discarded, and 400 μL of 0.1 M TRIS (pH 7.5) was added, followed by spinning at 14,000 rpm for another 30 minutes. This process was repeated once more. A 25 μL aliquot of trypsin solution (1 μg / mL) was then added to the sample, followed by 50 μL of 0.1 M TrisHCl (pH 7.5). After gentle mixing, the sample was placed in a 37°C incubator for overnight digestion (18–20 hours). 100 μL of 7.5 M guanidine-HCl (pH 5.0) was added to the sample, followed by gentle shaking for 30 minutes. All samples were spun at 14,000 rpm for 45 minutes using a Beckman Coulter microfuge 18 centrifuge and the flow-through (approximately 200 μL) was collected for LC / MS analysis.

[0294] LC A nanoLC system (EASY-nLC 1000, Thermo Fisher Scientific) was used to separate the tryptic peptides collected after sample preparation. An analytical column (75 μm × 250 mm, Thermo Scientific EASY-Spray™ HPLC) with a 2 μm particle size and a trap column (75 μm × 20 mm, Thermo Fisher Scientific Acclaim™ PepMap™ 100 C18 HPLC column) were used, with the trap column preceding the analytical column. Peptide digests were first loaded onto the trap column and then eluted from the trap column onto the analytical column for further separation. Mobile phase A was 0.1% formic acid / 99.9% water, and mobile phase B was 0.1% formic acid / 99.9% acetonitrile. The flow rate was 300 nL / min, and the column temperature was set at 40°C. A gradient of 3% to 25% B over 80 min was used.

[0295] MS For LC-MS analysis, this nanoLC system was coupled to a Q Exactive mass spectrometer (Thermo Scientific). The nanoflow source conditions were set as follows: spray voltage = 1.8 kV, transfer capillary temperature = 180 °C, and S-lens = 50 V. Full MS scans were acquired using profile data mode with a resolution of 70,000, an AGC target of 3E6, a maximum injection time of 120 ms, and an m / z scan range of 350–1800. MS / MS scans were acquired using centroid data with a resolution of 17,500, an AGC target of 1E5, and a maximum injection time of 200 ms. The top 10 most abundant peptide ions were selected for MS / MS analysis with dynamic exclusion over a 30-second period.

[0296] Data Processing MS data were processed using MassAnalyzer 4.10 to generate MGF files for Mascot database searches. The MS noise level was set to 3,000, with a minimum signal-to-noise ratio of 3.

[0297] Mascot Database search for HCP identification After generating a searchable file (MGF file) using MassAnalyzer, this was used for a Mascot database search to identify host cell proteins (HCPs). The search parameters used are listed below: peptide tolerance: 15 ppm, MS / MS tolerance: 0.02 Da or less, primary digestion reagent: trypsin, secondary digestion reagent: none, missed cleavages: 0, fixed modification reagent: carbamidomethyl C, and database: UniProt CHO 2013.

[0298] HCP quantification MassAnalyzer used the average peak area of ​​the top three most abundant peptides to represent the abundance or level of the protein from which the top three peptides were derived. HCP levels were calculated using the formula: HCP level (ng / mg) = average peak area of ​​the top three peptides of (HCP x HCP molecular weight) / (average peak area of ​​the top three peptides of antibody x antibody molecular weight).

[0299] In addition to antibodies, added yeast standard proteins glucose-6-phosphate dehydrogenase (G6PD) and inorganic pyrophosphatase (IPYR) were used to perform relative quantification of AEP and other host cell proteins.

[0300] result AEP characterization to understand mAb1 fragmentation The biomanufacturing process for mAb1 was designed to achieve high CHO cell densities during production cell culture to increase titer, however, drug substance and in-process sample material showed increased fragmentation at the N192 / F193 site (EU numbering) of the heavy chain (Figure 3).

[0301] In-process samples from a pilot-scale run of PSL1, which employed a target pH of 4.6 during acid precipitation and exhibited high levels of fragmentation, were used for AEP characterization. To understand the cause of fragmentation, nanoflow LC-MS / MS proteomics analysis was performed on the in-process samples. AEP was detected at high levels, particularly in the upstream sample, correlating with the observation that fragmentation occurs primarily in the upstream process (Figure 1). The levels of AEP in the upstream and downstream materials indicated that AEP was present at higher abundance (approximately 30-100-fold higher) before the downstream purification process, followed by removal of most AEP by the Protein A affinity purification step. Additionally, the level of fragmentation remained relatively constant for samples collected throughout the downstream process, and further storage in the pH 5.2 formulation buffer over an extended period did not increase fragmentation.

[0302] Further characterization of AEP demonstrated that activation from the proenzyme form to the mature form occurs during acid precipitation, and that fragmentation occurs primarily during this acid precipitation. 325 , D 305 , and E 311 CHO AEP peptide (LMSTN 325 DLK (SEQ ID NO: 1) and LD 305 LTPSPE 311 The intact peptides (LMSTNDDLK (SEQ ID NO: 1) and LDLTSPEVPLTILK (SEQ ID NO: 2)) reflected the proenzyme form of CHO AEP. After acid precipitation, the intensities of these peptides were reduced approximately 100-fold, indicating that AEP is a cleavage and activation indicator. 325 , D 305 , and E 311 The autocleavage site D was cleaved into the mature active form (Fig. 2), but the total AEP level remained the same before and after acid precipitation treatment. 25 and D 28 Since no AEP peptide containing this was detected, this AEP peptide was not used as an indicator of the AEP proenzyme.

[0303] In summary, to understand the cause of mAb1 fragmentation, AEP characterization of in-process samples of mAb1 was performed by nanoLC-MS. Results showed high levels of AEP in the upstream process and low levels in the downstream process. The AEP levels correlated well with the observation that the majority of fragmentation originated from the acid precipitation step performed at pH 4.6 in the upstream process. Four parameters appeared to affect the fragmentation of the IgG2 antibody mAb1: retention time; AEP concentration relative to mAb1 concentration; solution pH; and the concentration of the mature, active form of AEP after autocleavage of the proenzyme form.

[0304] Fragmentation induced by added AEP in the mAb1 reference standard To further confirm that AEP was the cause of mAb1 fragmentation, 400 ng of AEP per mg of antibody was added to the mAb1 reference standard, followed by overnight incubation at 37°C. This addition resulted in approximately 2.5% fragmentation at pH 4.6, but only approximately 0.5% fragmentation at pH 7.0 (Table 1). The level of fragmentation caused by the added AEP (2.5%, 400 ng / mg AEP, Table 1) qualitatively matched that observed in the process (4%, 1085 ng / mg AEP in the HCCF sample, Table 1).

[0305] [Table 1]

[0306] Characterization of PSL2 and PSL3 AEPs for evaluation of fragmentation control strategies To evaluate the effect of acid precipitation pH on AEP-induced fragmentation while still maintaining high recovery and downstream purification, two lots of mAb1 material were produced using different pHs for the acid precipitation procedure: AP pH 4.9 for lot PSL2 and AP pH 5.1 for lot PSL3. High levels of AEP in the upstream samples were detected in both lots (Table 2). However, PSL3 had very low levels of fragmentation, similar to that of the reference standard (as measured by rCE-SDS). Results demonstrated that increasing the pH used for the acid precipitation step to pH 5.1 for PSL3 significantly suppressed AEP enzymatic activity at pH 5.1, thereby reducing the level of AEP-induced fragmentation by inhibiting both AEP activation by autocleavage and its activity toward mAb1.

[0307] Characterization of AEP from PSL2 and PSL3 development lots showed that PSL3 had higher levels of total host cell protein (HCP) and AEP (due to cell culture variability) but lower clipping levels compared to PSL2 (Table 2). Adjusting the acid precipitation pH from 4.6 in PSL1 to 5.1 in PSL3 resulted in lower fragmentation levels. This indicated that despite the observed higher AEP levels in PSL3, adjusting the pH to 5.1 in the AP was highly effective in reducing clipping to RS levels (0.3%). No significant differences in downstream process performance were observed with respect to AEP removal; clearance rates were similar in the Protein A affinity chromatography step for both PSL2 and PSL3 (Table 2). Residual AEP levels were higher in PSL3 compared to PSL2 in the Protein A and DS steps, but the fragmentation levels observed in the DS were low and comparable to the RS material. The levels of AEP in the DS of PSL2 and PSL3 were also lower than those of PSL1 and closer to those of the RS, likely due to the additional purification step in the CEX step after Protein A (Table 2). These results demonstrate that increasing the pH to 5.1 during acid precipitation can be an effective fragmentation control strategy without other process modifications. Acid precipitation at pH 5.1 was still effective in removing cells, DNA, and other cellular debris (data not shown).

[0308] [Table 2]

[0309] Assessment of the effect of residual AEP on the DS stability of mAb1 PSL3 Because residual AEP was still carried over into the DS, the impact of AEP on mAb1 drug substance (DS) stability was evaluated using the DS from PSL3. The fragmentation levels observed in the stressed PSL3 DS were similar to those of the reference standard (RS) material from the stressed lot (Table 3), indicating that residual AEP does not significantly affect DS stability (relative to LMW by rCE-SDS). The aggregation rate in the SE-HPLC assay was also typical, suggesting that acid precipitation at pH 5.1 was still effective in removing cells, DNA, and other cellular debris.

[0310] [Table 3]

[0311] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and journal articles, are hereby expressly incorporated by reference. Any described embodiment of the present disclosure may be combined with one or more other embodiments of the present disclosure, unless the context clearly indicates otherwise.

[0312] The presently disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, but instead as non-limiting exemplifications of particular aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. Indeed, various modifications of the presently disclosed subject matter in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the presently disclosed subject matter.

[0313] The descriptions of various embodiments and / or examples of the subject matter of the present disclosure are presented for purposes of illustration and are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has been selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the marketplace, and / or to enable those skilled in the art to understand the subject matter of the present disclosure.

Claims

1. 1. A method for recovering a recombinant protein during a biomanufacturing process, comprising: establishing a cell culture by inoculating a bioreactor with mammalian cells expressing the recombinant protein; maintaining the cell culture during a growth phase and a production phase, optionally mixing the cell culture with an exogenous protease inhibitor during said growth phase and / or during or after said production phase; cooling the cell culture, optionally maintaining a dissolved oxygen level of about 64 mmHg to about 128 mmHg in the cell culture during said cooling; combining the cell culture with an acidic solution to obtain an acidified cell culture having a pH of about 4.6 to about 5.3; and incubating the acidified cell culture to induce aggregation of one or more cell culture impurities. Including, the recombinant protein comprises an asparaginyl endopeptidase (AEP) cleavage site; method.

2. 10. The method of claim 1, wherein the cell culture is admixed with an exogenous protease inhibitor during the growth phase, the production phase, and / or the cooling phase.

3. 3. The method of claim 1 or 2, wherein the exogenous protease inhibitor is an AEP inhibitor.

4. 4. The method of claim 1, wherein a dissolved oxygen level of about 64 mmHg to about 128 mmHg is maintained in the cell culture during the cooling.

5. The method of any one of claims 1 to 4, wherein the cell culture is cooled to about 4°C to about 15°C.

6. 6. The method of any one of claims 1 to 5, wherein the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and any combination of the foregoing.

7. 7. The method of any one of claims 1 to 6, comprising incubating the acidified cell culture for at least about 60 minutes.

8. 8. The method of any one of claims 1 to 7, further comprising separating the one or more cell culture impurities from the acidified cell culture by continuous solids discharge centrifugation, disc stack centrifugation, and / or depth filtration to obtain a clarified cell culture.

9. 9. The method of any one of claims 1 to 8, wherein the pH of the acidified cell culture is from about 4.9 to about 5.

2.

10. measuring the amount or activity of AEP prior to said mixing; and / or measuring the amount of AEP proenzyme prior to said mixing; The method of any one of claims 1 to 9, further comprising:

11. 1. A method for inhibiting cleavage of a recombinant protein containing an asparaginyl endopeptidase (AEP) cleavage site during a biomanufacturing process involving acid precipitation, comprising: Measuring the amount or activity of AEP and / or the amount of one or more fragments of the recombinant protein in a sample isolated from a cell culture operation, a harvesting operation, or a purification operation of the biomanufacturing process; and If the amount or activity of the AEP and / or the amount of one or more fragments thereof exceeds a threshold value, adjusting the pH of the acid precipitation to about 4.6 to about 5.

3. A method comprising:

12. 12. The method of claim 11, wherein cleavage of the recombinant protein is inhibited compared to recombinant protein produced by an alternative biomanufacturing process that uses an acid precipitation pH of less than about 4.

6.

13. 13. The method of claim 11 or 12, wherein the sample is isolated from a cell culture or harvesting operation.

14. measuring the amount of AEP using mass spectrometry or immunoassay; or The activity of the AEP is measured using a cleavage assay. The method according to any one of claims 11 to 13.

15. If the amount or activity of the AEP exceeds the threshold, adjusting the dissolved oxygen level of the cell culture to between about 64 mmHg and about 128 mmHg during a cell culture cooling operation of the biomanufacturing process; and / or If the amount or activity of the AEP exceeds the threshold, combining an exogenous protease inhibitor with the cell culture during the growth and / or production phase of the biomanufacturing process. The method of any one of claims 11 to 14, further comprising:

16. The threshold amount of AEP is greater than about 1000 ng of AEP per mg of harvested cell culture fluid (HCCF); and / or the AEP activity threshold exceeds about 3% clipping of the recombinant protein at the AEP cleavage site; and / or the threshold being at least about 1% w / w of the recombinant protein being the one or more fragments; The method according to any one of claims 11 to 15.

17. 17. The method of any one of claims 11 to 16, comprising adjusting the pH of the acid precipitation to about 4.9 to about 5.2 if the amount or activity of the AEP and / or the amount of the one or more fragments exceeds the threshold.

18. The method of any one of claims 1 to 17, wherein the recombinant protein comprises a N192 / F193 sequence motif in the CH1 domain of the heavy chain constant region according to EU numbering.

19. The method of any one of claims 1 to 18, wherein the recombinant protein is an IgG2 antibody.

20. 20. The method of any one of claims 1 to 19, wherein the biomanufacturing process comprises a perfusion cell culture process, an enriched fed-batch cell culture process, or an intensive cell culture process.

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