Commercial scale recombinant protein production in rat hybridoma cells

Abstract

The present disclosure relates to methods of making recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells (e.g., YB2 / 0) with high productivity, product quality, and robust functional activity by modifying cell culture process parameters and / or basal medium and feed combinations. The disclosure also relates to commercial scale production (e.g., 10,000 L to 25,000 L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells. The disclosure also relates to recombinant proteins (e.g., monoclonal antibodies) and compositions (e.g., medicaments) that are made using the methods disclosed herein and have unique glycosylation profiles.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 347,793, filed on June 1, 2022. The entire contents of the foregoing are hereby incorporated by reference.

[0003] Citation of electronically submitted sequence listing

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named "50581-0003WO1.XML." This XML file was created on May 22, 2023 and is 19,157 bytes in size. The material in this XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure is in the field of mammalian cell culture for the production of recombinant proteins (e.g., monoclonal antibodies). More specifically, the present disclosure is in the field of commercial scale production (e.g., 10,000L-25,000L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells. Background Art

[0006] Recombinant proteins (e.g., antibodies) are becoming increasingly important as therapeutic agents for a wide range of diseases such as cancer, autoimmune diseases, and infectious diseases. See, Kaplon, H. et al., MAbs 11:219-238 (2019); Lu, RM. et al., Journal of Biomedical Science 27:1 (2020). Worldwide, at least 570 therapeutic mAbs have been studied in clinical trials, and as of December 2019, 79 therapeutic mAbs have been approved by the U.S. Food and Drug Administration (FDA) and are currently on the market, including 30 mAbs for the treatment of cancer. Ibid.

[0007] In most cases, therapeutic proteins (e.g., antibodies) are produced in cell culture by mammalian cells that have been engineered and / or selected to produce high levels of the polypeptide of interest. In fact, among all approved recombinant protein-based biopharmaceuticals, mammalian cells dominate over other recombinant protein expression systems (Owczarek, B. et al., BioMed Research International 2019: Article ID 4216060, 1-13 (2019). Chinese hamster ovary (CHO) cells, mouse hybridoma cells (NS0), and mouse hybridoma (Sp2 / 0) cells are the main mammalian cell lines used to express recombinant biopharmaceuticals, with CHO-based systems contributing the largest percentage (about 84%) (Tripathi, NK et al., Front Bioeng Biotechnol. 7: 420 (2019)).

[0008] Rat hybridoma cell lines have also been used (although at much lower frequencies than other cell lines) for the production of therapeutic antibodies. For example, rat hybridoma cell line YB2 / 0 has been used to produce anti-Rh (D) monoclonal antibodies with enhanced antibody-dependent cellular cytotoxicity (ADCC) function, which can be used to prevent Rh blood group system alloimmunization (Rhesus isoimmunization) in Rh-negative individuals. See, U.S. Patent Nos. 7,931,895 and 8,409,572 assigned to LFB Biotechnologies. U.S. Patent No. 9,234,045 assigned to Laboratoire Francais du Fractionnement et desBiotechnologies relates to monoclonal antibodies directed against CD20 antigen, which are produced by rat hybridoma YB2 / 0 cell lines in addition to other cell lines. These patents are incorporated herein by reference in their entirety. However, it is still necessary to produce monoclonal antibodies in rat hybridoma cells on a commercial scale.

[0009] In mammalian cell culture, medium formulation, cell line, cell culture and process control parameters can potentially affect the resulting recombinant protein yield and product quality profile. See, Li, F. et al., Mabs 2: 466-477 (2010). Characterization and understanding of the effects of culture process parameters such as culture pH, dissolved CO2 (pCO2), temperature, culture medium, etc. on process performance and product quality attributes are the main reasons why these relationships are queried during process characterization studies and tracked during good manufacturing process (GMP) manufacturing. In addition, a relationship with one cell line does not necessarily apply to another cell line. Jiang, R. et al., Bioprocess Biosyst Eng 41: 1731-1741 (2018). The process parameter acceptance limit is defined by the manufacturer of the therapeutic protein to ensure that the product with the desired specifications is always achieved.

[0010] Protein glycosylation is a post-translational modification (PTM) that can affect the product quality of recombinant proteins. Asparagine-linked (N-linked) glycosylation is very common on recombinant therapeutic glycoproteins (especially antibodies). N-linked protein glycosylation is usually composed of 5 major sugars ( Figure 1 ), and has been shown to be decisive for the physiochemistry, pharmacokinetics, immunogenicity, and Fc effector functions of the protein to which it is attached. Therefore, protein glycosylation (e.g., fucosylation) is often classified as a critical quality attribute (CQA) during bioprocessing and is closely monitored during manufacturing to ensure compliance with sponsor-defined acceptable limits.

[0011] Optimization of mammalian cell culture process parameters is important for successful, cost-effective and reproducible commercial production of recombinant proteins with desired specifications, increased titer, high product quality profile (e.g., low fucosylation) and robust biological activity. Therefore, there is a need to improve both the productivity and product quality of recombinant proteins in mammalian cell expression systems, particularly for therapeutic monoclonal antibodies produced on a commercial scale. Summary of the invention

[0012] Provided herein are methods for manufacturing recombinant proteins (e.g., monoclonal antibodies) in a mammalian expression system that is not commonly used, rat hybridoma cell lines (e.g., YB2 / 0). Also provided are methods for increasing product titer and improving the product quality and functional activity of these recombinant proteins by modifying cell culture process parameters and / or cell culture medium. Also provided are methods for commercial-scale production (e.g., 10,000L-25,000L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells that maintain high protein quality and functional activity. In particular aspects, the methods of the present disclosure are used to produce anti-CD20 antibodies on a commercial scale. Also provided herein are compositions manufactured according to the methods of the present disclosure, including pharmaceutical compositions. In some aspects, the compositions manufactured according to the methods of the present disclosure exhibit unique glycosylation patterns, which contribute to consistent product quality, clinical safety, and efficacy.

[0013] Thus, provided herein is a method for producing at least 10,000 L of an antibody protein in a rat hybridoma cell by culturing the rat hybridoma cell in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cell comprises an expression vector comprising a polynucleotide encoding the antibody protein.

[0014] In some embodiments, the culture pH is about 6.5 to about 7.0. In some embodiments, the culture pH of about 6.5 to about 7.0 is set on culture day 2 of the cell culture. In some embodiments, the culture pH of about 6.5 to about 7.0 is set on culture day 3 of the cell culture.

[0015] In some embodiments, the culture pH is about 7.0 to about 7.55. In some embodiments, the culture pH of about 7.0 to about 7.55 is set on culture day 0 to day 3 of the cell culture.

[0016] In some embodiments, the culture pH is reduced to about 6.5 to about 7.0 on day 2 or day 3 of cell culture. In some embodiments, the culture pH is reduced on day 3 of cell culture. In some embodiments, the culture pH is maintained at about 6.5 to about 7.0 from culture day 3 of cell culture until harvest.

[0017] In some embodiments, the cumulative culture time and cumulative decrease (integrated pH2 difference) allowed for the pH to drop below the fixed pH set point after the pH is reduced on day 3 are smaller relative to cell cultures with a larger integrated pH2 difference. In some embodiments, the fixed pH set point is pH 6.91.

[0018] In some embodiments, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and a higher titer at harvest. In some embodiments, a lower integrated pH2 difference further results in a lower fucosylation percentage.

[0019] In some embodiments, rat hybridoma cells expressing antibody proteins are cultured in a chemically defined animal component-free (ADCF) culture medium.

[0020] In some embodiments, the harvest titer of the antibody protein is increased and / or the fucosylation of the antibody protein is reduced when the culture pH is 6.6 to 6.96, relative to cell culture under the same culture conditions except that the culture pH is 6.60 to 6.8.

[0021] In some embodiments, the method further comprises controlling the culture pCO2 level to less than about 300 mmHg. In some embodiments, the pCO2 level of less than about 300 mmHg is promoted by supplementing the cell culture with additional buffer, increasing the air sparging rate, increasing the dissolved oxygen (DO) set point, and / or reducing the agitation rate.

[0022] In some embodiments, the method further comprises an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1. In some embodiments, the method further comprises a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3.

[0023] In some embodiments, the end of culture day 1 is 17 to 33 hours after initiating the cell culture.

[0024] In some embodiments, the method further comprises a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest. In some embodiments, the third temperature set point is 32.5°C.

[0025] In some embodiments, the cell culture comprises the following culture conditions: i) an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0026] In some embodiments, the production of the antibody protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150% relative to the antibody protein produced by a culture process that does not employ the culture conditions described herein.

[0027] In some embodiments, the method further comprises harvesting the antibody protein produced by the rat hybridoma cells.

[0028] In some embodiments, the method further comprises purifying the antibody protein by affinity chromatography and / or ion exchange chromatography. In some embodiments, the affinity chromatography comprises protein A purification. In some embodiments, the purified antibody protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation. In some embodiments, the quality of the purified antibody protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF), and / or N-linked glycan analysis.

[0029] In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (eg, a monoclonal antibody) is an anti-CD20 antibody.

[0030] In some embodiments, the monoclonal antibodies are subjected to CD20, FcγRIIIa-158V, and / or C1q binding assays.

[0031] In some embodiments, the monoclonal antibody has a relative potency of 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, CD20 binding is determined by binding of the anti-CD20 antibody to the human mantle cell lymphoma cell line Jeko-1 expressing CD20.

[0032] In some embodiments, the percentage of FcγRIIIa-158V binding is about 82% to about 130% relative to a commercial reference standard used in the binding assay.In some embodiments, the percentage of FcγRIIIa-158V binding is determined by surface plasmon resonance (SPR).

[0033] In some embodiments, the monoclonal antibody has a relative potency of 86% to 117% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some embodiments, the monoclonal antibody has a relative potency of 88% to 113% in a C1q binding assay as measured by ELISA compared to a commercial reference standard.

[0034] In some embodiments, the monoclonal antibody has a relative potency of 74% to 127% in a cell-based complement dependent cytotoxicity (CDC) assay compared to a commercial reference standard.

[0035] In some embodiments, the monoclonal antibody produced has a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity relative to the monoclonal antibody produced by the culture process without the culture conditions described herein. In some embodiments, the monoclonal antibody has a relative efficacy of 90% to 163% in cell-based ADCC assays compared to commercial reference standards. In some embodiments, the monoclonal antibody has a relative efficacy of about 117% in cell-based ADCC assays compared to commercial reference standards.

[0036] In some embodiments, the monoclonal antibody comprises: a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7 and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7 and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7 and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0037] In some embodiments, the monoclonal antibody comprises a deletion of up to 5 N-terminal residues. In some embodiments, the monoclonal antibody comprises a deletion of up to 10 N-terminal sequences.

[0038] In some embodiments, the cell culture is carried out in a bioreactor. In some embodiments, the bioreactor is a commercial scale bioreactor. In some embodiments, the commercial scale bioreactor is a 10,000L, 15,000L, 20,000L or 25,000L bioreactor. In some embodiments, the commercial scale bioreactor is a 15,000L bioreactor.

[0039] In some embodiments, the rat hybridoma cell is a YB2 / 0 rat hybridoma cell.

[0040] Also provided herein is a method for producing antibody proteins in culture of rat hybridoma cells at a commercial scale, the method comprising the steps of: a) preparing and thawing a working rat hybridoma cell bank of the antibody protein of interest; b) expanding the size and volume of the culture of rat hybridoma cells from the cell bank through a series of shake flasks (125 mL, 500 mL, 3 L, 3x3 L shake flasks, and 50 L cell bags) with a target seeding density of at least 0.30 x 10 6 viable cells / mL; c) process the cell culture through a series of seed bioreactors (120L, 600L and 3,000L) to further increase the volume and cell culture mass; d) inoculate the cell culture from the 3,000L seed bioreactor into the commercial scale production bioreactor; e) harvest the cell culture supernatant from the commercial scale production bioreactor; f) clarify the recovered cells by serial centrifugation followed by depth filtration; g) purify the antibody protein by protein A capture column chromatography; and h) inactivate viral agents by solvent detergent viral inactivation (SDVI).

[0041] In some embodiments, the commercial scale production bioreactor is operated in fed-batch mode.

[0042] In some embodiments, a drilled hole (10) gas injector having an orifice diameter of 4.0 mm is used in a commercial scale production bioreactor.

[0043] In some embodiments, the method further comprises purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX).

[0044] In some embodiments, the method further comprises viral filtration (VF) to remove potential viruses.

[0045] In some embodiments, the method further comprises ultrafiltration / diafiltration (UFDF).

[0046] In some embodiments, the method further includes preparing a bulk drug formulation containing an antibody protein by adding polysorbate 80 to the formulation buffer to prepare a bulk drug substance formulation. In some embodiments, the method further includes subjecting the bulk drug formulation to 0.2 μm filtration. In some embodiments, the method further includes filling a 6L bag to a target filling volume of 5.50L for the bulk drug formulation, and storing the bulk drug formulation at ≤-35°C. In some embodiments, the antibody protein in the bulk drug formulation is formulated into a pharmaceutically acceptable formulation. In some embodiments, the method further includes testing the microorganisms and viral adventitious agents of the unprocessed bulk harvest from commercial-scale production bioreactors. In some embodiments, the method further includes removing and / or inactivating microorganisms and viral adventitious agents from commercial-scale production bioreactors.

[0047] In some embodiments, the rat hybridoma cell is a YB2 / 0 cell.

[0048] In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (eg, a monoclonal antibody) is an anti-CD20 antibody.

[0049] In some embodiments, the monoclonal antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0050] In some embodiments, the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0051] In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0052] In some embodiments, the antibody protein comprises an N-glycan profile comprising one or both of: i) about 10 to 20% galactosylated glycans; and / or ii) about 20 to 40% fucosylated glycans.

[0053] In some embodiments, the N-glycan profile includes about 10 to 20% galactosylated glycans and about 23% to 36% fucosylated glycans. In some embodiments, the N-glycan profile includes about 23% to about 36% fucosylated glycans. In some embodiments, the N-glycan profile includes about 16% to about 18% galactosylated glycans. In some embodiments, the N-glycan profile includes about 17% galactosylated glycans.

[0054] In some embodiments, the antibody protein comprises an N-glycan profile comprising at least about 10% bisecting N-glycans. In some embodiments, the N-glycan profile comprises about 12% to about 30% bisecting N-glycans. In some embodiments, the N-glycan profile comprises about 18% bisecting N-glycans.

[0055] In some embodiments, the antibody protein comprises an N-glycan profile comprising less than 5% sialylated glycans. In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some embodiments, the N-glycan profile comprises an undetectable amount of sialylated glycans.

[0056] In some embodiments, the antibody protein comprises an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans. In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, Man5 N-glycans are the only high mannose species in the N-glycan profile.

[0057] In some embodiments, the antibody protein is produced at a commercial scale of about 10,000 L to about 25,000 L. In some embodiments, the commercial scale is 15,000L.

[0058] In some embodiments, the methods produce a harvest titer of antibody protein of about 0.5 g / L to about 1.5 g / L. In some embodiments, the harvest titer is about 1.0 g / L to about 1.5 g / L.

[0059] Also provided herein are antibody proteins made according to the methods described herein. In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (e.g., a monoclonal antibody) is an anti-CD20 antibody.

[0060] The present invention also provides a rat hybridoma master cell bank (MCB) composition comprising antibody proteins having at least two of the following parameters: i) about 11 to about 13 x 10 6 cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 3% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoforms as detected by iCIEF; and / or x) about 20% to about 36% basic isoforms as detected by iCIEF.

[0061] Also provided herein are rat hybridoma working cell bank (WCB) compositions comprising antibody proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 2% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoforms as detected by iCIEF; and / or x) about 14% to about 38% basic isoforms as detected by iCIEF.

[0062] In some embodiments, the rat hybridoma cells in the cell bank are YB2 / 0 cells. In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (e.g., a monoclonal antibody) is an anti-CD20 antibody.

[0063] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0064] In some embodiments, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0065] Also provided herein are methods of making antibody proteins by using the MCB or WCB compositions described herein.

[0066] In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (eg, a monoclonal antibody) is an anti-CD20 antibody.

[0067] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0068] In some embodiments, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0069] In some embodiments, the method for manufacturing a recombinant protein in a rat hybridoma cell comprises culturing a rat hybridoma cell in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cell comprises an expression vector comprising a polynucleotide encoding the recombinant protein. In some aspects, the culture pH is about 6.5 to about 7.0. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the second day of the culture of the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the third day of the culture of the cell culture. In some aspects, the culture pH is about 7.0 to about 7.55. In some aspects, the culture pH of about 7.0 to about 7.55 is set at the 0th to the 3rd day of the culture of the cell culture.

[0070] In some embodiments, the culture pH is reduced to about 6.5 to about 7.0 on day 2 or day 3 of cell culture. In some embodiments, the culture pH is reduced on day 3 of cell culture. In some embodiments, the culture pH is maintained at about 6.5 to about 7.0 from culture day 3 of cell culture until harvest.

[0071] In some embodiments, relative to the cell culture with a larger integral pH2 difference, the cumulative culture time and cumulative decline (integrated pH2 difference) that allow pH to drop below the fixed pH set point after the pH is reduced on the 3rd day are smaller. In some aspects, the fixed pH set point is pH 6.91. In some aspects, a lower integral pH2 difference results in a higher integral viable cell density (IVCD) and a higher titer at harvest. In some aspects, a lower integral pH2 difference further results in a lower fucosylation percentage.

[0072] In some embodiments, rat hybridoma cells expressing the recombinant protein are cultured in chemically defined animal component-free (ADCF) medium.

[0073] In some embodiments, the harvest titer of the recombinant protein is increased and / or the fucosylation of the recombinant protein is reduced when the culture pH is 6.6 to 6.96 relative to cell culture under the same culture conditions except that the culture pH is 6.60 to 6.8.

[0074] In some embodiments, the method of making a recombinant protein in a rat hybridoma cell further comprises controlling the culture pCO2 level to be less than about 300 mmHg. In some aspects, the pCO2 level of less than about 300 mmHg is promoted by supplementing the cell culture with additional buffer, increasing the air sparging rate, increasing the dissolved oxygen (DO) set point, and / or reducing the agitation rate.

[0075] In some embodiments, the method of producing a recombinant protein in rat hybridoma cells further comprises an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1. In some aspects, the method of producing a recombinant protein in rat hybridoma cells further comprises a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3. In some aspects, the end of culture day 1 is 17 to 33 hours after starting cell culture.

[0076] In some embodiments, the method of producing recombinant protein in rat hybridoma cells further comprises a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest. In some aspects, the third temperature set point is 32.5°C.

[0077] In some embodiments, the cell culture in the methods disclosed herein includes the following culture conditions: i). an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest; ii). a culture pH between about 6.5 and about 7.55; and iii). a culture pCO2 of less than about 300 mmHg. In some aspects, the yield of the recombinant protein produced by the methods disclosed herein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150% relative to a recombinant protein produced by a culture process that does not employ the culture conditions in i), ii), and iii).

[0078] In some embodiments, the method for manufacturing a recombinant protein in a rat hybridoma cell further comprises harvesting the recombinant protein produced by the rat hybridoma cell. In some aspects, the method further comprises purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some aspects, the affinity chromatography comprises protein A purification.

[0079] In some embodiments, the purified recombinant protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation.

[0080] In some embodiments, the quality of the purified recombinant protein produced by the methods disclosed herein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis. In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody specifically binds to an epitope of CD20.

[0081] In some embodiments, the monoclonal antibody undergoes CD20, FcγRIIIa-158V and / or C1q binding assays. In some aspects, the monoclonal antibody has a relative efficacy of 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some aspects, CD20 binding is determined by the binding of an anti-CD20 antibody to a human mantle cell lymphoma cell line Jeko-1 expressing CD20. In some aspects, relative to a commercial reference standard for binding assays, the percentage of FcγRIIIa-158V binding of the monoclonal antibody is about 82% to about 130%. In some aspects, the percentage of FcγRIIIa-158V binding is determined by surface plasmon resonance (SPR). In some aspects, compared to commercial reference standards, as measured by ELISA, the monoclonal antibody has a relative efficacy of 86% to 117% in a C1q binding assay. In some aspects, the monoclonal antibody has a relative potency of 88% to 113% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some aspects, the monoclonal antibody has a relative potency of 74% to 127% in a cell-based complement dependent cytotoxicity (CDC) assay compared to a commercial reference standard. In some aspects, relative to a monoclonal antibody produced by a culture process that does not employ the following culture conditions: i) an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg, the produced monoclonal antibody has a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity. In some aspects, the monoclonal antibody has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some aspects, the monoclonal antibody has a relative potency of about 117% in a cell-based ADCC assay compared to a commercial reference standard.

[0082] In some embodiments, the monoclonal antibody produced by the methods disclosed herein comprises: a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7 and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7 and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7 and a light chain having the amino acid sequence shown in SEQ ID NO: 9. In some aspects, the monoclonal antibody comprises a deletion of up to 5 N-terminal residues. In some aspects, the monoclonal antibody comprises a deletion of up to 10 N-terminal sequences.

[0083] In some embodiments, the cell culture is carried out in a bioreactor. In some aspects, the bioreactor is a commercial scale bioreactor. In some aspects, the commercial scale bioreactor is a 10,000L, 15,000L, 20,000L or 25,000L bioreactor. In some aspects, the commercial scale bioreactor is a 15,000L bioreactor.

[0084] In some embodiments, the rat hybridoma cell expressing the recombinant protein is a YB2 / 0 rat hybridoma cell.

[0085] Also provided herein are recombinant proteins made according to any of the methods disclosed herein.In some aspects, the recombinant protein is a monoclonal antibody.

[0086] Also provided herein is a method for producing a recombinant protein in culture of rat hybridoma cells at a commercial scale, the method comprising the steps of: a) preparing and thawing a working rat hybridoma cell bank of a recombinant protein of interest; b) expanding the size and volume of the culture of rat hybridoma cells from the cell bank through a series of shake flasks (125 mL, 500 mL, 3 L, 3x3 L shake flasks, and 50 L cell bags) with a target seeding density of at least 0.30 x 10 6viable cells / mL; c) processing the cell culture through a series of seed bioreactors (120L, 600L and 3,000L) to further increase the volume and cell culture quality; d) inoculating the cell culture from the 3,000L seed bioreactor into the commercial scale production bioreactor; e) harvesting the cell culture supernatant from the commercial scale production bioreactor; f) clarifying the recovered cells by continuous centrifugation followed by depth filtration; g) purifying the recombinant protein by protein A capture column chromatography; and h) inactivating viral material by solvent detergent viral inactivation (SDVI). In some aspects, the commercial scale production bioreactor is operated in fed-batch mode. In some aspects, a drilled (10) gas sparger with a 4.0 mm orifice diameter is used in the commercial scale production bioreactor.

[0087] In some embodiments, the method for commercial scale production of recombinant protein further includes purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX). In some aspects, the method further includes virus filtration (VF) to remove potential viruses. In some aspects, the method further includes ultrafiltration / diafiltration (UFDF). In some aspects, the method further includes preparing bulk bulk drug formulations containing recombinant protein, including adding polysorbate 80 in the formulation buffer to prepare bulk bulk drug formulations. In some aspects, the method further includes subjecting the bulk bulk drug formulations to 0.2 μm filtration. In some aspects, the method further includes filling 6L bags to a target filling volume of 5.50L for bulk bulk drug formulations, and storing the bulk bulk drug formulations at ≤-35 ° C. In some aspects, the recombinant protein in the bulk bulk drug formulation is formulated into a pharmaceutically acceptable formulation. In some aspects, the method further includes testing the microorganisms and viral foreign substances of the untreated bulk harvest from commercial scale production bioreactors. In some aspects, the method further comprises removing and / or inactivating microbial and viral extraneous material from the commercial scale production bioreactor.

[0088] In some embodiments, in methods for commercial-scale production of recombinant proteins, the rat hybridoma cells are YB2 / 0 cells.

[0089] In some embodiments, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody binds to an epitope of CD20. In some aspects, the monoclonal antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0090] In some embodiments, the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0091] In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:8.

[0092] In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:9.

[0093] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile comprising one or both of the following: i) about 10 to 20% galactosylated glycans; and / or ii) about 20 to 40% fucosylated glycans. In some embodiments, the N-glycan profile comprises about 10 to 20% galactosylated glycans and about 20 to 40% (e.g., about 23 to 36%) fucosylated glycans. In some embodiments, the N-glycan profile comprises about 23% to about 36% fucosylated glycans. In some embodiments, the N-glycan profile comprises about 16% to about 18% galactosylated glycans. In some embodiments, the N-glycan profile comprises about 17% galactosylated glycans.

[0094] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile comprising at least about 10% bisecting N-glycans. In some embodiments, the N-glycan profile comprises about 12% to about 30% bisecting N-glycans. In some embodiments, the N-glycan profile comprises about 18% bisecting N-glycans.

[0095] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile comprising less than 5% sialylated glycans. In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycans. In some embodiments, the N-glycan profile comprises an undetectable amount of sialylated glycans.

[0096] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans. In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some aspects, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, Man5 N-glycans are the only high mannose species in the N-glycan profile.

[0097] In some embodiments, the recombinant protein or monoclonal antibody is produced at a commercial scale of about 10,000 L to about 25,000 L. In some embodiments, the commercial scale is 15,000L.

[0098] In some embodiments, the disclosed methods produce a recombinant protein or monoclonal antibody harvest titer of about .5 g / L to about 1.5 g / L. In some embodiments, the harvest titer is about 1.0 g / L to about 1.5 g / L.

[0099] Also provided herein are rat hybridoma master cell bank (MCB) compositions and rat hybridoma working cell bank (WCB) compositions that can be used to produce the recombinant proteins (eg, monoclonal antibodies) disclosed herein.

[0100] In some embodiments, the rat hybridoma MCB provided herein comprises recombinant proteins having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all of the following parameters: i) about 11 to about 13 x 10 6cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 3% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoforms as detected by iCIEF; and / or x) about 20% to about 36% basic isoforms as detected by iCIEF.

[0101] In some embodiments, the rat hybridoma MCB provided herein comprises recombinant proteins having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all of the following parameters: i) about 11 to about 13 x 10 6 cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 3% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoforms as detected by iCIEF; and / or x) about 20% to about 36% basic isoforms as detected by iCIEF.

[0102] In some embodiments, the rat hybridoma WCB provided herein comprises recombinant proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 2% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoforms as detected by iCIEF; and / or x) about 14% to about 38% basic isoforms as detected by iCIEF.

[0103] In some embodiments, the rat hybridoma WCB provided herein comprises recombinant proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 2% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoforms as detected by iCIEF; and / or x) about 14% to about 38% basic isoforms as detected by iCIEF.

[0104] In some embodiments, the rat hybridoma cells in the MCB composition or WCB composition are YB2 / 0 cells. In some embodiments, the recombinant protein to be produced from the MCB or WCB composition is a monoclonal antibody. In some embodiments, the monoclonal antibody is an anti-CD20 antibody.

[0105] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:9.

[0106] Also provided are methods for making recombinant proteins by using an MCB or WCB composition as disclosed herein. In some aspects, the recombinant protein is a monoclonal antibody. In some embodiments, the monoclonal antibody is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Describes nitrogen-linked (N-linked) protein glycosylation species ("N-glycans") that can be covalently attached to the recombinant proteins described herein and can be released and quantified by enzymatic deglycosylation. N-glycans generally include five major sugars: N-acetyl-D-glucosamine (GlcNAc); mannose (Man); galactose (Gal); fucose (Fuc); and N-acetylneuraminic acid (NANA), as shown in the figure.

[0108] Figure 2 is a bar graph showing the effect of different cell culture media on process performance - cell growth (as measured by IVCD), as described in Example 1. The numbers 1105-1152 along the X-axis reflect the different cell culture media. Table 5 provides the name of the cell culture media associated with a given number.

[0109] Figure 3 is a bar graph showing the effect of different cell culture media on the process performance - Cell Viability, as described in Example 1. The numbers 1105-1152 along the X-axis reflect the different cell culture media. Table 5 provides the name of the cell culture media associated with a given number.

[0110] Figure 4 is a bar graph showing the effect of different cell culture media on process performance - harvest titer, as described in Example 1.

[0111] Figure 5 is a bar graph showing the effect of different cell culture media on the process performance: % fucosylation, as described in Example 1.

[0112] Figure 6 is a scatter plot showing the effect of changes in cell culture medium on % fucosylation, as described in Example 1.

[0113] Figure 7 is a scatter plot showing the effect of changes in cell culture medium on FcγRIIIa-158V binding, as described in Example 1.

[0114] Figure 8 is a scatter plot showing the effect of changes in cell culture medium on CD20 binding, as described in Example 1.

[0115] Fig. 9 is a scatter plot showing the effect of changes in cell culture medium on ADCC activity, as described in Example 1.

[0116] Fig.10 is a scatter plot showing the effect of changes in cell culture medium on C1q binding, as described in Example 1.

[0117] Fig.11 is a scatter plot showing the effect of changes in cell culture medium on CDC activity, as described in Example 1.

[0118] Fig.12 is a scatter plot showing cumulative cell growth (IVCD) versus cumulative pCO2 exposure, as described in Example 2.

[0119] Fig.13 is a scatter plot showing harvest titer versus cumulative pCO2 exposure.

[0120] Fig.14 is a graph showing the viable cell density (VCD) results of cell cultures evaluating different pH control ranges (post-shift), as described in Example 2.

[0121] Fig.15 is a graph showing the results of evaluating cell viability (%) of cell cultures at different pH control ranges (after shift), as described in Example 2.

[0122] Fig.16 is a graph showing in-process glucose results for cell culture evaluating different pH control ranges (after transition), as described in Example 2.

[0123] Fig.17 is a graph showing in-process lactate results for cell culture evaluating different pH control ranges (after transition), as described in Example 2.

[0124] Fig.18 is a graph showing the in-process pCO2 results for cell culture evaluating different pH control ranges (after transition), as described in Example 2.

[0125] Fig.19 is a graph showing the in-process titer results for evaluating cell culture at different pH control ranges (after shift), as described in Example 2.

[0126] Figure 20 depicts a map of expression vector HK463-25 containing immunoglobulin heavy and light chain cDNA sequences of the source anti-CD20 antibody TG-1101 described herein.

[0127] Fig.21 is a diagram schematically depicting the process parameter "Integrated pH2 Difference", as described in Example 5.

[0128] Fig. 22is a graph depicting the variability of post-shift pH ("pH2") in a typical process C2 culture, as described in Example 5.

[0129] Fig.23 is a graph depicting the effect of integrated pH2 differences on IVCD, as described in Example 5.

[0130] Fig.24 is a graph depicting the effect of integrated pH2 differences on harvest titers, as described in Example 5.

[0131] Fig.25 is a graph depicting the effect of integrated pH 2 differences on percent fucosylation, as described in Example 5.

[0132] Fig.26 The glycosylation profiles of samples of anti-CD20 antibodies were described as described in Example 6. DETAILED DESCRIPTION

[0133] Provided herein are methods for manufacturing recombinant proteins (e.g., monoclonal antibodies) in uncommon mammalian expression systems, rat hybridoma cells (e.g., YB2 / 0). Also provided are methods for increasing product titer and improving reproducibility, homogeneity, product quality (e.g., low fucosylation percentage), and functional activity of recombinant proteins by modifying cell culture process parameters and / or cell culture medium. Also provided are methods for commercial-scale production (e.g., 10,000 L, 15,000 L, 20,000 L, 25,000 L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells that maintain high protein quality and functional activity. Also provided herein are compositions manufactured according to the methods of the present disclosure, including pharmaceutical compositions.

[0134] In some embodiments, the methods disclosed herein are used to produce anti-CD20 antibodies at a commercial scale (e.g., 10,000L-25,000L). In some aspects, the anti-CD20 antibodies manufactured by the manufacturing process described herein have a unique glycosylation profile, which will be further described below. Without being bound by theory, the relative distribution of various N-glycans or individual sugar residues present in these N-glycans can determine the biological and clinical properties of anti-CD20 antibodies. See, co-pending and co-owned U.S. Provisional Application No. 63 / 347,852, entitled “Anti-CD20 Antibody Compositions,” filed on June 1, 2022, which is incorporated herein by reference in its entirety.

[0135] abbreviation

[0136] Table 1 provides a list of abbreviations used in this disclosure.

[0137] Table 1: List of abbreviations

[0138]

[0139]

[0140] definition

[0141] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning shown below. Additional definitions are shown throughout the application.

[0142] The term "and / or" as used herein should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0143] It is to be understood that whatever aspects are described herein with the language "comprising," other similar aspects described with the terms "consisting of" and / or "consisting essentially of are also provided.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure to the skilled person.

[0145] Units, prefixes and symbols are expressed in the form recognized by the International System of Units (SI). Numerical ranges include numbers that limit the range. The headings provided herein are not limitations of the various aspects of the disclosure, and various aspects of the disclosure can be obtained by quoting the entire specification. Therefore, the terms defined immediately below are more fully defined by quoting the specification in its entirety.

[0146] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a or an" should be understood to mean "more than one" of any listed or enumerated components.

[0147] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth. Generally, unless otherwise indicated, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10% up or down (higher or lower).

[0148] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and (where appropriate) include fractions thereof (such as tenths and hundredths of integers).

[0149] As used herein, the term "inoculating" or "seeding" refers to the process of providing a cell culture to a bioreactor (e.g., a production bioreactor) or another container. In some aspects, the cells have been previously propagated in another bioreactor or container. In some aspects, the cells have been frozen and thawed immediately before being provided to the bioreactor or container.

[0150] As used herein, the term "cell culture" refers to growing mammalian cells (e.g., rat hybridoma cells) in suspension in a culture medium according to the present disclosure. As will be apparent from the context, the term "cell culture" or "in cell culture" may also refer to a closed container, vessel, or bioreactor used to grow mammalian cells. See also "bioreactor."

[0151] As used herein, the term "bioreactor" refers to a commercial scale, large-scale, small-scale, or microscale container for growing mammalian cells (e.g., rat hybridoma cells) according to the methods disclosed herein. Bioreactors allow "control" of various cell culture parameters during cell culture processes, including but not limited to circulation loop flow, pH, temperature, overpressure, and / or medium perfusion rate. Bioreactors include, for example, commercially available bioreactors, stirred tank bioreactors, airlift bioreactors, bubble column bioreactors, hollow fiber bioreactors, fluidized bed bioreactors, membrane bioreactors, classic fermenters, benchtop bioreactors, 10-15 ml and 250 ml microscale bioreactors (e.g., ambr (Sartorius)) and cell culture perfusion systems, and disposable or single-use bioreactors.

[0152] The bioreactor can be any size useful for culturing cells at a desired scale according to the methods of the present disclosure. For example, in some aspects, the bioreactor employed in the methods of the present disclosure can range from about 250 ml to about 25,000 liters. In some aspects, the volume of the bioreactor is about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160 , 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 3 30, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500 0, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,0000, 13,000, 14,000, 15,000, 20,000 liters, 25,000 liters or more, or any intermediate volume.

[0153] In some aspects, recombinant protein production is at commercial scale. In some aspects, commercial scale bioreactors can be 10,000L to 25,000L. Therefore, as used herein, the term "commercial scale production" of a recombinant protein (e.g., an antibody) refers to production in at least 10,000L, at least 15,000L, at least 20,000L, or at least 25,000L bioreactors. In some aspects, a commercial scale bioreactor is 15,000L.

[0154] Suitable bioreactors can be made of any material suitable for maintaining cell culture under the culture conditions disclosed herein and conducive to cell growth and viability. For example, the bioreactors used in the methods disclosed herein can be made of glass, plastic or metal. Suitable bioreactors are known in the art and are commercially available.

[0155] As used herein, the term "production bioreactor" refers to any container made of glass, plastic or metal, between 15ml and 25,000L, supporting a culture environment conducive to cell growth. For example, in Example 3, the production bioreactor is a 15,000L stainless steel bioreactor. In some respects, such as described in Example 3, the term "production bioreactor" (or N culture container) refers to a series of final bioreactors in containers (such as N-4, N-3, N-2 and N-1 containers or "seed bioreactors") that increase scale, which is inoculated with a cell culture (also referred to as "seed culture (seed culture)") from the N-1 container immediately before it with a high viable cell density, and cells will continue to grow therein until harvest. In some respects, the cells in cell culture are harvested from the production bioreactor. (see, for example, Example 3).

[0156] As used herein, the term "seed bioreactor" refers to any cell culture vessel used prior to transferring the cell culture to a production bioreactor. (See, e.g., Example 3).

[0157] As used herein, the terms "control," "controlling," or "controlled" refer to the ability to purposefully increase, decrease, or maintain the same protein structure / function parameter. In the methods of the present disclosure, culture pH and temperature are examples of controllable cell culture parameters.

[0158] As used herein, the term "cell" refers to mammalian cells, cultured cells, host cells, recombinant cells and recombinant host cells. Such cells are typically cell lines obtained or derived from mammalian tissues that can grow and survive when placed in a culture medium containing appropriate nutrients and / or growth factors. The cells utilized in the methods of the present disclosure are mammalian rat hybridoma cells (e.g., YB2 / 0) that can express and secrete, or that can be molecularly engineered to express or secrete a large amount of recombinant proteins (e.g., antibodies) into the culture medium.

[0159] Typically, "cells" as described herein are cultured for a continuous number of "culture days" (or, alternatively, "process days") until they are harvested. In some aspects, the number of "culture days" can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days or more.

[0160] As used herein, "culture day 1" refers to about 12.0 hours to about 35.9 hours after the cells in the seed culture are inoculated into the production bioreactor. As used herein, "culture day 2" refers to about 36.0 hours to about 59.9 hours after the cells in the seed culture are inoculated into the production bioreactor. As used herein, "culture day 3" refers to about 60.0 hours to about 83.9 hours after the cells in the seed culture are inoculated into the production bioreactor.

[0161] As used herein, the term "culture condition(s)" refers to cell culture conditions, such as pH, temperature, pCO2 at various levels described herein or shifted among these levels, which result in the expression of a recombinant protein (e.g., an antibody) with increased growth, titer, cell density, cell viability, or improved product quality relative to a cell culture not cultured under the specific culture condition(s).

[0162] As used herein, the terms "media," "culture media," "cell culture media," and "basal media," including grammatical variations thereof, are used interchangeably and refer to the physiochemical, nutritional, and hormonal environment in which mammalian cells (e.g., rat hybridoma cells) can grow in culture and express a recombinant protein of interest (e.g., a monoclonal antibody). Exemplary cell culture media are described below.

[0163] As used herein, the term "setting" (e.g., "setting" pH) refers to inputting a specific value (or values) of a process parameter (such as, for example, temperature, pH) into a bioreactor or other cell culture vessel control system. See also "set point".

[0164] As used herein, unless otherwise indicated, "set point" refers to the setting of conditions for a bioreactor or other cell culture container for growing cells and / or producing a protein product. In some aspects, the set point is set on a production bioreactor. The set point can be established at the beginning of cell culture and / or reset to a different set point during cell culture. For example, in some aspects, the set point can be a "pH set point". In some aspects, the set point is a "temperature set point". In some aspects, the set point can be maintained throughout the cell culture method. In other aspects, the set point can be maintained until a different set point is set. In other aspects, the set point can be changed to another set point.

[0165] As used herein, "temperature set point" refers to the temperature setting of a bioreactor (e.g., a production bioreactor) or other cell culture process container for growing cells and / or producing a protein product. The temperature set point can be established at the beginning of the cell culture in the production bioreactor, where it may also be referred to as the "initial temperature set point". The subsequent temperature change during the cell culture after the initial temperature set point is referred to as the second temperature set point, or the subsequent third temperature set point. The last temperature set point before harvesting may also be referred to as the "final temperature set point". In some aspects, a process may include an initial temperature set point, a second temperature set point, and a third (and final) temperature set point.

[0166] In some aspects, the "initial temperature set point" is set at culture day 0 to culture day 1. In some aspects, the "second temperature set point" is set at the end of culture day 1 to culture day 3. In some aspects, the "third temperature set point" is set at culture day 3 and maintained until harvest.

[0167] As used herein, the term "pH 1" refers to the culture pH in the production bioreactor after cells in the seed culture are inoculated into the production bioreactor.

[0168] As used herein, the term "pH 2" refers to the culture pH in the production bioreactor after the pH shifts from the culture pH after inoculation ("pH 1") to a lower pH on culture day 3 (about 62 to 77 hours, preferably 72 hours).

[0169] As used herein, the term "integrated pH2 difference" is a calculated pH parameter that refers to the cumulative culture time and cumulative decline in pH allowed to drop below the fixed pH set point after the pH is reduced on the third day of culture. When the culture pH drops below the fixed pH set point for any time measurement unit (unit measure of time), the integrated pH2 difference increases. In some aspects, relative to cell cultures with larger "integrated pH2 differences", the integrated pH2 difference of cell cultures involving fixed pH set points (e.g., 6.91) is smaller. In some aspects, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD), a higher titer at harvest, and a lower percentage of fucosylation.

[0170] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. The term refers to the primary structure of a molecule. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Polynucleotides may be recombinantly, enzymatically or synthetically manufactured, for example by solid phase chemical synthesis followed by purification. When referring to the sequence of a polynucleotide or nucleic acid, reference is made to the sequence or order of the covalently linked nucleobase moieties of the nucleotides or their modifications.

[0171] As used herein, the terms "protein," peptide, and "polypeptide" are used interchangeably to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. Also included in the definition are, for example, polypeptides containing more than one analog of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that since the polypeptides of the present invention are antibody-based, in some aspects, the polypeptides may appear as single chains or associated chains.

[0172] As used herein, "recombinant protein" refers to a polypeptide or protein produced via recombinant DNA technology. As disclosed herein, recombinantly produced polypeptides and proteins are expressed in engineered host cells (such as, for example, rat hybridoma cells). The term "protein" is intended to include glycoproteins.

[0173] As used herein, the term "percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by comparing the two sequences, introducing gaps to maximize the identity between the sequences. Alignment can be generated using programs known in the art. For purposes of this article, the alignment of nucleotide sequences can be performed using the blastn program set to default parameters, and the alignment of amino acid sequences can be performed using the blastp program set to default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov).

[0174] As used herein, the term "expression vector" refers to any nucleic acid construct containing the necessary elements (e.g., promoters, enhancers) for transcription and translation of an inserted coding sequence for a polypeptide of interest when introduced into a host cell (e.g., a rat hybridoma cell). Expression vectors may include plasmids, phages, viruses, and derivatives thereof. Expression vectors of the present disclosure may include polynucleotides encoding recombinant proteins (e.g., monoclonal antibodies).

[0175] As used herein, the term "glycoprotein" refers to a protein modified by the addition of one or more carbohydrate moieties (e.g., polysaccharides or oligosaccharides) linked to the protein via oxygen- or nitrogen-containing side chains of amino acid residues, such as serine or threonine residues ("O-linked") or asparagine residues ("N-linked").

[0176] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide composed of monosaccharide residues, such as a polymer or an oligomer.

[0177] As used herein, in the context of protein (eg, antibody) production, the term "upstream processes" refers to activities involved in the production and collection of proteins (eg, antibodies) from cells (eg, during cell culture of recombinant proteins).

[0178] As used herein, in the context of protein (e.g., antibody) preparation, the term "downstream process" refers to one or more techniques used after upstream process techniques in order to purify the protein (e.g., antibody) of interest. For example, downstream process techniques include purification of protein products using, for example, affinity chromatography including protein A affinity chromatography, ion exchange chromatography such as anion or cation exchange chromatography, virus filtration, depth filtration, ultrafiltration, diafiltration, and centrifugation.

[0179] As used herein, the term "glycosylation" associated with recombinant glycoproteins refers to the addition of complex oligosaccharide structures to proteins at specific sites within the polypeptide chain. Glycosylation of proteins and subsequent processing of added carbohydrates can affect protein folding and structure, protein stability (including protein half-life) and the functional properties of proteins. Protein glycosylation can be divided into two categories, O-linked glycosylation and N-linked glycosylation, by virtue of the sequence context in which the modification occurs. O-linked polysaccharides are connected to hydroxyl groups, usually to the hydroxyl group of any one of serine or threonine residues. O-glycans are not added to every serine and threonine residue. O-linked oligosaccharides are usually monoantennary or biantennary, that is, they contain one or at most two branches (antennas), and contain one to four different types of sugar residues added one by one. N-linked polysaccharides are connected to the amide nitrogen of asparagine. Only asparagine that is part of one of two tripeptide sequences, either asparagine-X-serine or asparagine-X-threonine (where X is any amino acid except proline), is a target for glycosylation. N-linked oligosaccharides can have from one to four branches, known as mono-, di-, tri-, or tetra-antennary. The structures of N- and O-linked oligosaccharides and the sugar residues found therein differ. Despite this difference, the terminal residues on each branch of both N- and O-linked polysaccharides can be modified with sialic acid residues. Sialic acid is the common name for a unique family of nine-carbon monosaccharides that can be linked to other oligosaccharides. The two main types of sialic acid residues found in biopharmaceuticals produced in mammalian expression systems are N-acetyl-neuraminic acid (NANA) and N-glycolyl-neuraminic acid (NGNA).

[0180] As used herein, the term "sialylation" refers to the addition of sialic acid (S) residues, e.g., N-glycans: G1FS1, G2S1, G2FS1, G2FBS1, G2S2, G2FS2, G2FBS2, to recombinant glycoproteins.

[0181] As used herein, the term "fucosylation" or "protein fucosylation" refers to the addition of fucose (F) residues, e.g., N-glycans: G0F-GN, G0F, G0FB, G1F, G1FB, and G2F, to recombinant glycoproteins.

[0182] As used herein, the term "galactosylation" refers to the addition of galactose (gal) residues, eg, N-glycans: G1, G2, G1F, G1FB, G2F, to a recombinant glycoprotein.

[0183] As used herein, the term "fucosylation percentage" or "fucosylation %" refers to the percentage of N-glycans with fucose among all N-glycans. Similarly, the term "galactosylation percentage" or "galactosylation %" refers to the percentage of N-glycans with galactose among all N-glycans. For example, the fucosylation percentage is calculated by subjecting a sample or population of anti-CD20 antibody proteins to enzymatic deglycosylation so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans among N-glycans cleaved using enzymatic digestion.

[0184] As used herein, the term "cell density" refers to the number of cells in a given volume of medium. Cell density can be monitored by any technique known in the art, including, but not limited to, extracting a sample from the culture and analyzing the cells under a microscope, using a commercially available cell counting device, or by using a commercially available suitable probe introduced into the bioreactor itself (or into a loop through which the culture medium and suspended cells pass and then return to the bioreactor).

[0185] As used herein, the term "integrated viable cell density" or "IVCD" refers to the viable cell density integrated relative to the duration of cell culture.

[0186] As used herein, the term "viable cell density" or "VCD" refers to the number of viable cells present in a given volume of culture medium under a given set of experimental conditions.

[0187] As used herein, the term "cell viability" refers to the ability of cells in a cell culture to survive under a given set of conditions or experimental variations. As used herein, the term also refers to the percentage (%) of cells that survive at a particular time relative to the total number of cells (e.g., live and dead) in the culture at that time.

[0188] As used herein, the term "shift" refers to a modulation or change in a particular cell culture parameter (eg, pH shift, temperature shift).

[0189] As used herein, the term "after the shift" (when before a specific value or percentage) indicates a value or percentage of a specific level (e.g., glucose, lactate, % fucosylation, % sialylation, titer) or activity (e.g., binding or effector function) of a cell culture or expressed recombinant protein (e.g., antibody) taken at any time after the shift in a specific cell culture parameter.

[0190] As used herein, the "initial growth phase" of cell culture refers to the period from day 0 to day 2 (i.e., day 0, 1, and / or day 2) during which cells (e.g., YB2 / 0) begin to grow. In the "initial growth phase," the amount of recombinant protein expressed by cell culture is significantly lower than in the subsequent protein production phase (i.e., day 3 to harvest).

[0191] As used herein, the "protein production phase" of cell culture refers to culture day 3 to harvest, during which rat hybridoma cells (eg, YB2 / 0) produce recombinant protein in significantly higher amounts than in the initial growth phase, so that the titer can be measured.

[0192] As used herein, the term "harvest" refers to the time point in a mammalian cell culture process when cells containing a recombinant protein are separated and removed from the cell culture medium and undergo additional processing (such as, for example, centrifugation, filtration, or purification). In some aspects, the harvest of cells will occur on process day 12 or day 13 of the cell culture, or when cell viability drops below 20%, whichever occurs first. See also, "harvest titer".

[0193] As used herein, the term "harvest material" refers to cells containing the recombinant protein that are recovered from a production bioreactor at the time of "harvest."

[0194] As used herein, the term "master cell bank" or "MCB" refers to a cell bank generated under GMP conditions from cells expanded from the TG-1101 producing YB2 / 0 cell line described herein.

[0195] As used herein, the term "working cell bank" or "WCB" refers to a cell bank generated under GMP conditions from cells expanded from an MCB.

[0196] As used herein, the term "bulk drug substance formulation" refers to the final formulated material at the end of the purification unit operation of the manufacturing process described herein.

[0197] As used herein, the term "unprocessed bulk" refers to harvest material from a production bioreactor prior to clarification.

[0198] As used herein, the term "harvest clarification" refers to the primary purification stage for removing cells, particles and impurities from the production bioreactor harvest material. See, e.g., Example 3. After the harvest clarification stage, the clarified harvest can be stored, e.g., for 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or ≤ 11 days.

[0199] As used herein, the term "single batch" in the context of a recombinant protein (e.g., an anti-CD20 antibody) refers to a composition derived from a single production or run of a single bioreactor of a specified volume. For example, an anti-CD20 antibody obtained from a single run of a 15,000L bioreactor can be referred to as a single batch. In some aspects, the anti-CD20 antibody is present in such a single batch at a concentration of at least 10 mg / ml; 15 mg / ml; 20 mg / ml; 25 mg / ml; or at least 30 mg / ml. In some aspects, the anti-CD20 antibody is present in such a single batch at a concentration between 10 and 35 mg / ml; 10 and 30 mg / ml; 10 and 25 mg / ml; 10 and 20 mg / ml; 10 and 15 mg / ml; 15 and 35 mg / ml; 15 and 30 mg / ml; 15 and 25 mg / ml; 15 and 20 mg / ml; 20 and 35 mg / ml; 20 and 30 mg / ml; 20 and 25 mg / ml; 25 and 35 mg / ml; or 25 and 30 mg / ml. In some aspects, the anti-CD20 antibody is present in such a single batch at a concentration of about 15 mg / ml; about 20 mg / ml; about 25 mg / ml; about 30 mg / ml; or about 35 mg / ml. In some aspects, the amount of total protein is quantified by spectrophotometry. In some aspects, the amount of total protein is quantified by spectrophotometric absorbance at 280 nm.

[0200] As used herein, the term "foreign material" refers to microorganisms (eg, bacteria, fungi, viruses, mycoplasmas) that can pose a risk to human health and are inadvertently introduced into the manufacturing process of biopharmaceuticals.

[0201] As used herein, the term "titer" refers to the total amount of recombinantly expressed proteins (e.g., antibodies) produced by cell culture divided by a given amount of culture medium volume. The term "titer" refers to concentration, and is usually described in milligrams (mg) of protein per milliliter (mL) or per liter (L) of culture medium. In some aspects, the methods of the present disclosure can significantly increase protein product titers compared to protein product titers produced by other cell culture methods known in the art, or by cell culture methods that do not employ culture conditions described herein.

[0202] As used herein, the term "harvest titer" refers to the total amount of protein (e.g., antibody) produced by cell culture when cells are harvested from a cell culture, cell culture vessel, or bioreactor. In some aspects, the harvest of cells will occur on process day 12 or day 13 of the cell culture, or when cell viability drops below 20%, whichever occurs first.

[0203] As used herein, the term "antibody" or "Ab" shall include, but is not limited to, a glycoprotein immunoglobulin or an antigen-binding portion thereof that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (referred to herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into highly variable regions called complementary determining regions (CDRs) interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0204] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art, and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0205] As an example, the term "antibody" includes monoclonal antibodies; polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. In the absence of explicit indication and unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or antigen-binding portions of any of the above-mentioned immunoglobulins, and includes monovalent and divalent fragments or portions, and single-chain antibodies.

[0206] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition (i.e., antibody molecules that are substantially identical in primary sequence and exhibit a single binding specificity and affinity for a particular epitope). A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma, recombination, transgenic, or other techniques known to those skilled in the art.

[0207] As used herein, the term "epitope" refers to a local region in an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, a combination of two or more non-continuous regions of one or more polypeptides (conformational, non-linear, discontinuous or non-continuous epitopes). Epitopes formed by continuous amino acids are usually (but not always) retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. An epitope typically includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or continuous peptides from (e.g., from L1CAM) are tested for reactivity with a given antibody (e.g., anti-L1CAM antibody). Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as x-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)). In some aspects, an antibody may bind to more than one epitope (e.g., TG-1101).

[0208] "HuMAb" refers to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0209] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. On the one hand, in a humanized form of an antibody, some, most or all of the amino acids outside the CDR have been replaced with amino acids from human immunoglobulin, while some, most or all of the amino acids in more than one CDR are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are allowed as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized antibody" retains an antigenic specificity similar to that of the original antibody.

[0210] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0211] As used herein, "acceptable range" or "AR" refers to the range of values ​​of certain process parameters (e.g., pH) whereby control within these values ​​ensures that process performance and product quality attributes meet their specifications. Deviations from the "acceptable range" may lead to formal root cause analysis and impact assessment.

[0212] Method for producing recombinant proteins (such as monoclonal antibodies) in rat hybridoma cells

[0213] In one aspect, the present disclosure provides a method for manufacturing a recombinant protein (e.g., a monoclonal antibody) in a rat hybridoma cell, the method comprising culturing cells in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cell comprises an expression vector comprising a polynucleotide encoding the recombinant protein. In some aspects, the culture pH is about 6.5 to about 7.0. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the second day of the culture of the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the third day of the culture of the cell culture. In some aspects, the culture pH is about 7.0 to about 7.55. In some aspects, the culture pH is set to about 7.0 to about 7.55 on the 0th to the 3rd day of the culture of the cell culture.

[0214] In some aspects, the culture pH is reduced to about 6.5 to about 7.0 on day 2 or day 3 of cell culture. In some aspects, the culture pH is reduced on day 3 of cell culture. In some aspects, the culture pH is maintained at about 6.5 to about 7.0 from culture day 3 of the cell culture until harvest.

[0215] "Integrated pH2 difference" is a calculated pH parameter, which refers to the cumulative culture time and cumulative decline in pH allowed to drop below the fixed pH set point after pH is reduced on the 3rd day of cultivation. When the culture pH drops below the fixed pH set point for any time measurement unit, the integrated pH2 difference increases. In some aspects, relative to the larger cell culture of "integrated pH2 difference", the integrated pH2 difference of the cell culture involving the fixed pH set point is smaller. In some aspects, the fixed pH set point is pH 6.91. In some aspects, the lower integrated pH2 difference leads to a higher integrated viable cell density (IVCD) and a higher titer when harvested. In some aspects, the lower integrated pH2 difference further leads to a lower fucosylation percentage.

[0216] In some aspects, rat hybridoma cells expressing recombinant proteins are cultured in a chemically defined medium free of animal derived components (AFCP). In some aspects, the basal culture medium is (Cytiva), feed medium was BalanCD CHO Feed (Irvine Scientific).

[0217] In some aspects, the harvest titer of the recombinant protein is increased and / or the fucosylation of the recombinant protein is reduced when the culture pH is 6.6 to 6.96 relative to cell culture under the same culture conditions except that the culture pH is 6.60 to 6.8.

[0218] In some aspects, the pCO2 level in the cell culture is controlled to be less than about 300 mmHg. In some aspects, the pCO2 level of less than about 300 mmHg is promoted by supplementing the cell culture with additional buffer, increasing the air sparging rate, increasing the dissolved oxygen (DO) set point and / or reducing the agitation rate.

[0219] In some aspects, the culture conditions further include an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1. In some aspects, the culture conditions further include a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3. In some aspects, "the end of culture day 1" is 17 to 33 hours after starting cell culture. In some aspects, the culture conditions further include a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest. In some aspects, the third temperature set point is 32.5°C.

[0220] In some aspects, the present disclosure relates to a method for manufacturing a recombinant protein, wherein the cell culture comprises the following culture conditions: i). an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest; ii). a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg. In some aspects, "the end of culture day 1" is 17 to 33 hours after starting cell culture.

[0221] In some aspects, the yield of the recombinant protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, relative to a recombinant protein produced by a culture process that does not employ: i) an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0222] In some aspects, the method of the present disclosure further comprises harvesting the recombinant protein produced by rat hybridoma cells. In some aspects, the purification of the recombinant protein is carried out by affinity chromatography and / or ion exchange chromatography. In some aspects, the affinity chromatography comprises protein A purification.

[0223] In some aspects, the purified recombinant protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation. In some aspects, the quality of the purified recombinant protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis.

[0224] In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody specifically binds to the epitope of CD20. In some aspects, the monoclonal antibody undergoes various assays to assess efficacy and biological activity. In some aspects, wherein the monoclonal antibody undergoes CD20, FcγRIIIa-158V and / or C1q binding assays. The results of these bioassays will be discussed below.

[0225] Rat hybridoma cells

[0226] Many mammalian cells or cell types that are susceptible to cell culture and expression of polypeptides are known in the art, such as, for example, BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblastoma cells (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells ± DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocarcinoma line (Hep G2).

[0227] In some aspects, the mammalian cell used to culture and express a recombinant protein (e.g., a monoclonal antibody) according to the methods of the present disclosure is a rat hybridoma cell. Exemplary rat hybridoma cells include YB2 / 0, IR983F, IR2, and IR162.

[0228] In some aspects, the rat hybridoma cells used in the methods of the present disclosure are YB2 / 0 rat hybridoma cells (ATCC CRL 1662).

[0229] Recombinant proteins (e.g. therapeutic antibodies)

[0230] Any protein or polypeptide that can be expressed in rat hybridoma cells can be produced as a recombinant protein according to the methods of the present disclosure. In one aspect, the recombinant protein that can be produced by rat hybridoma cells according to the present disclosure includes, for example, any pharmaceutically or commercially relevant antibody, enzyme, receptor, hormone, regulatory factor, antigen or binding agent.

[0231] Worldwide, at least 570 therapeutic monoclonal antibodies (mAbs) have been studied in clinical trials by commercial companies, and as of December 2019, 79 therapeutic mAbs have been approved by the U.S. FDA and are currently on the market, including 30 mAbs for the treatment of cancer. And there is still significant growth potential. See Lu, R.-M. et al., Journal of Biomedical Science 27:1 (2020). The top 10 best-selling therapeutic antibody drugs in 2018 include Rituximab (Rituxan): anti-CD20 for non-Hodgkin lymphoma; Trastuzumab (Herceptin): anti-HER2 for breast cancer; Infliximab (Remicade): anti-TNFα for Crohn's disease; Pembrolizumab (Keytruda): anti-PD-1 for melanoma; Nivolumab (Opdivo): anti-PD-1 for melanoma and non-small cell lung cancer. Ustekinumab (Stelara): anti-IL-12 / 23 for psoriasis; Eculizumab (Soliris): anti-C5 for paroxysmal nocturnal hemoglobinuria; Bevacizumab (Avastin): anti-VEGF for colorectal cancer (CRC); Omalizumab (Xolair): anti-IgE for asthma; and Adalimumab (Humira): anti-TNFα for rheumatoid arthritis (RA). Same as above.

[0232] In view of the large number of antibodies currently in use or under study as therapeutic agents, the production of antibodies according to the methods of the present disclosure is a preferred aspect. Any antibody or its antigen binding fragment that can be produced in rat hybridoma cells can be used according to the present disclosure. In some aspects, the antibody to be produced is a monoclonal antibody or its antigen binding fragment. In some aspects, the antibody is a polyclonal antibody or its antigen binding fragment. In some aspects, the antibody is a chimeric antibody. In some aspects, the antibody is a humanized antibody. In some aspects, the antibody is a human antibody.

[0233] In some aspects, the above-mentioned monoclonal, polyclonal, chimeric or humanized antibodies may contain amino acid residues that do not naturally occur in any antibody of any species in nature. These exogenous residues can be utilized, for example, to confer novel or modified specificity, affinity or effector function to the monoclonal, chimeric or humanized antibodies.

[0234] Anti-CD20 antibody

[0235] In one aspect, the monoclonal antibodies produced according to the methods of the present disclosure bind to CD20 or an epitope of CD20. The term "anti-CD20 antibody" or "antibody that binds to CD20 or an epitope of CD20" refers to an antibody that can bind to CD20 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting CD20. The degree of binding of the anti-CD20 antibody to unrelated non-CD20 proteins is less than about 10% of the binding of the antibody to CD20, as measured by, for example, radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD20 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

[0236] CD20 is a hydrophobic transmembrane phosphoprotein expressed primarily in pre-B cells and mature peripheral B cells in humans and mice. In humans, CD20 is also strongly and homogeneously expressed in most mature B cell malignancies, including, for example, most non-Hodgkin's B cell lymphomas (NHL) and B-type chronic lymphocytic leukemia (B-CLL). The CD20 antigen is not expressed on hematopoietic stem cells or plasma cells. Anti-CD20 monoclonal antibodies have been developed and continue to be developed for the treatment of B cell diseases including B cell malignancies.

[0237] Chimeric anti-CD20 monoclonal antibody rituximab It has become the standard of care for many CD20-positive B-cell lymphomas and is the first mAb approved for any oncology indication. Demarest, SJ et al., mAbs 3:338-351 (2011). Biosimilars of rituximab are now FDA-approved, including rituximab-abbs (TRUXIMA) and rituximab-pvvr RITUXAN for subcutaneous use (rituximab and human hyaluronidase) injection was approved by the FDA in 2017.

[0238] In addition to rituximab, many other anti-CD20 antibodies are known in the art, including, for example, ublituximab (TG-1101), ofatumumab (HuMax; Intracel), ocrelizumab, veltuzumab, GA101 (obinutuzumab), AME-133v (Applied Molecular Evolution), ocaratuzumab (Mentrik Biotech), PRO131921, tositumomab, ibritumomab-tiuxetan, hA20 (Immunomedics, Inc.), BLX-301 (Biolex Biotech), Therapeutics), Reditux (Dr. Reddy's Laboratories), and PRO70769 (described in WO2004 / 056312).

[0239] Rituximab is a genetically engineered chimeric mouse / human monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody referred to as "C2B8" in U.S. Patent No. 5,736,137. The amino acid sequence of rituximab and an exemplary method of producing it via recombinant expression in CHO cells are disclosed in U.S. Patent No. 5,736,137, which is incorporated herein by reference in its entirety. Rituximab was initially approved by the FDA in 1997 for the treatment of non-Hodgkin's lymphoma.

[0240] Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody. Studies have shown that ofatumumab dissociates from CD20 at a slower rate than rituximab and binds to a membrane proximal epitope. Zhang et al., Mabs 1:326-331 (2009). Epitope mapping showed that ofatumumab binds to an epitope located closer to the N-terminus of CD20 than the position targeted by rituximab, and includes the extracellular loops of the antigen. Ibid.

[0241] As used herein, "TG-1101" (TG Therapeutics, Inc.) (also known as urituximab, UBX, UTX, TG-1101, TGTX-1101, Utuxin TM , LFB-R603, TG20, EMAB603) are the source antibodies for the anti-CD20 antibodies described herein having unique glycosylation profiles produced by the methods of the present disclosure.

[0242] Source antibody TG-1101 is a monoclonal antibody targeting epitopes on CD20 such as IRAHT (SEQ ID NO: 16) and EPAN (SEQ ID NO: 17). See, Fox, E. et al., Mult. Scler. 27: 420-429 (March 2021); Babiker et al., Expert Opin Investig Drugs 27: 407-412 (2018); Cotchett, KR et al., Multiple Sclerosis and Related Disorders 49: 102787 (2021); Miller et al., Blood 120: Abstract No. 2756 (2012); Deng, C. et al., J. Clin. Oncol. 31: Abstract No. 8575 (2013). TG-1101 is also described in U.S. Patent Nos. 9,234,045 and 9,873,745.

[0243] TG-1101 has been studied in a variety of cancer patient populations (e.g., NHL, CLL) both as a single agent and in combination with other agents. For example, O'Connor, OA et al., J. Clin. Oncol. 32:5s (2014), (Supplement; Abstract No. 8524), showed that TG-1101 was well tolerated and active in patients exposed to rituximab (rituxin). In a Phase I trial, Lunning, M. et al., American Society of Hematology Annual Meeting and Exhibition, December 5-8, 2015, Abstract No. 1538, showed that TG-1101 and TGR-1202 exhibited activity and a favorable safety profile in relapsed / refractory B-cell NHL and high-risk CLL. And in a Phase II trial, Sharman J. et al., American Society of Hematology (ASH) Annual Meeting and Exposition, December 5-8, 2015, Abstract No. 3980, showed that the combination of TG-1101 and Ibrutinib was highly active in patients with relapsed and / or refractory mantle cell lymphoma. The results of several studies involving the combination of TG-1101 with other agents (e.g., umbralisib, TG-1701, venetoclax) have been reported at the ASH Annual Meeting (December 5-8, 2020), publications 543, 3137, and 1130.

[0244] In addition, TG-1101 has been studied in Phase 2 and Phase 3 clinical trials to treat relapsing forms of multiple sclerosis (RMS). See, e.g., Fox, E. et al., Mult. Scler. 27:420-429 (March 2021) Steinman, L. et al., Neurology 96: (Suppl. 15) 4494 (2021). See also, pending U.S. Provisional Application No. 63 / 303,267 filed on January 26, 2022 and pending U.S. Provisional Application No. 63 / 288,350 filed on December 10, 2021, which are incorporated by reference in their entirety.

[0245] The amino acid (AA) and nucleotide sequences of antibody TG-1101 (TG Therapeutics, Inc.) are shown in Table 11.

[0246] Table 11: Sequence Listing of Source Anti-CD20 Antibody TG-1101

[0247]

[0248]

[0249]

[0250] In some aspects, TG-1101 comprises the VH CDR1, CDR2, and CDR3 regions of sequences SEQ ID NOs: 1, 2, and 3, and the VL CDR1, CDR2, and CDR3 regions of sequences SEQ ID NOs: 4, 5, and 6.

[0251] In some aspects, TG-1101 comprises a heavy chain (HC) that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7 and a light chain (LC) that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0252] In some aspects, TG-1101 comprises the HC of SEQ ID NO:7 and the LC of SEQ ID NO:8.

[0253] In some aspects, TG-1101 comprises the HC of SEQ ID NO:7 and the LC of SEQ ID NO:9.

[0254] In some aspects, the monoclonal antibodies produced by the methods disclosed herein comprise: a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; and a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0255] In some aspects, the monoclonal antibodies produced by the methods disclosed herein comprise a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:8.

[0256] In some aspects, the monoclonal antibodies produced by the methods disclosed herein comprise a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7 and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0257] In some aspects, the monoclonal antibody produced by the methods disclosed herein comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:8.

[0258] In some aspects, the monoclonal antibody produced by the methods disclosed herein comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:9.

[0259] In some aspects, the monoclonal antibodies produced by the methods disclosed herein comprise a deletion of up to 5 N-terminal residues.

[0260] In some aspects, the monoclonal antibodies produced by the methods disclosed herein comprise deletions of up to 10 N-terminal sequences.

[0261] In some aspects, the monoclonal antibody produced by the methods disclosed herein comprises the VH of SEQ ID NO:10 and the VL of SEQ ID NO:12.

[0262] In some aspects, the monoclonal antibodies produced by the methods disclosed herein bind to the same epitope as TG-1101 (TG Therapeutics, Inc.). In some aspects, the monoclonal antibodies produced by the methods disclosed herein are anti-CD20 antibodies (ie, bind to an epitope of CD20).

[0263] Transfection of the expression vector into rat hybridoma cells

[0264] Nucleic acids sufficient to achieve expression (usually an expression vector containing a gene encoding a polypeptide or protein of interest and any operably linked genetic control elements) are introduced into rat hybridoma cells by a number of well-known techniques. As used herein, the term "transfection" refers to the introduction of one or more exogenous polynucleotides (e.g., antibodies) into rat hybridoma cells by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol. 7: 2031-2034 (1987)).

[0265] For example, as described in Example 3, the expression vector HK463-25 (see FIG. 20 ) containing the immunoglobulin heavy and light chain cDNA sequences of TG-1101 (TG Therapeutics, Inc.) was transfected into YB2 / 0 host cells to produce the anti-CD20 antibody TG-1101 in a 15,000 L production bioreactor.

[0266] The cells are screened to determine which rat hybridoma cells actually take up the vector and express the polypeptide or protein of interest. Conventional methods for detecting a particular polypeptide or protein of interest expressed by mammalian cells include, but are not limited to, immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, SDS-PAGE, Western blotting, enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) techniques, bioactivity assays, and affinity chromatography.

[0267] Rat hybridoma cell culture

[0268] Once the cell expressing the polypeptide or protein of interest is identified, the cell is propagated in culture by any of the various methods well known to those skilled in the art. The cell expressing the protein of interest is usually propagated by growing it in a culture medium at a temperature conducive to the survival, growth and vitality of the cell. The initial culture volume can be any size, but is often less than the culture volume of the production bioreactor used in the final production of the protein of interest, and the cell is often passaged several times in a bioreactor of increased volume before seeding the production bioreactor. The cell culture can be stirred or shaken to increase the oxygenation of the culture medium and the dispersion of nutrients to the cells. Alternatively or additionally, special injection devices well known in the art can be used to increase and control the oxygenation of cell culture. According to the present disclosure, it will be appreciated by those of ordinary skill in the art that it may be advantageous to control or regulate certain internal conditions of the bioreactor, including but not limited to pH, temperature, oxygenation, etc.

[0269] The starting cell density in the production bioreactor can be selected by one of ordinary skill in the art. According to the present disclosure, the starting cell density in the production bioreactor can be as low as a single cell per culture volume. In some aspects, the starting cell density in the production bioreactor can range from about 0.1 x 10 per mL to about 0.1 x 10 per mL. 6 to about 10x 10 6 In some aspects, the starting cell density in the production bioreactor can range from about 0.1 x 10 6 About 2.0x 10 6 In some aspects, the starting cell density in the production bioreactor can be 2×10 2 , 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 , 5×10 6 , or 10×10 6 living cells and higher.

[0270] Before seeding the next mid-term or final production bioreactor, initial and mid-term cell culture can grow to any desired density. Although total viability or near total viability is not required, it is preferred that most cells remain alive before seeding. In one embodiment of the present disclosure, cells can be removed from the supernatant, for example, by low-speed centrifugation. It can also be desirable to wash the removed cells with culture medium before seeding the next bioreactor to remove any unwanted metabolic waste or culture medium components. The culture medium can be the culture medium in which the cells were previously grown, or can be different culture mediums or cleaning solutions selected by the implementer of the present disclosure.

[0271] The cells can then be diluted to an appropriate density for seeding a production bioreactor. In a preferred aspect of the present disclosure, the cells are diluted into the same culture medium that will be used in the production bioreactor. Alternatively, the cells can be diluted into another culture medium or solution, for example if they are to be stored for a short time before seeding a production bioreactor, depending on the needs and expectations of the implementer of the present disclosure, or in order to accommodate the specific requirements of the cells themselves.

[0272] As described above, once the production bioreactor has been seeded, the cell culture enters the "initial growth phase". In certain aspects, the "initial growth phase" refers to the culture days 0 to 2 (i.e., day 0, day 1, and / or day 2) of the cell culture, during which the cells (e.g., YB2 / 0) begin to grow. In the "initial growth phase", the amount of recombinant protein expressed by the cell culture is significantly lower than the "protein production phase" (i.e., culture day 3 to harvest) of the cell culture. The exact conditions will vary according to the cell type, the organism from which the cell is derived, and the properties and characteristics of the recombinant protein expressed.

[0273] After the "initial growth phase" is the "protein production phase" of cell culture. In certain aspects, the "protein production phase" refers to the period from day 3 of culture to harvest, during which cells (e.g., YB2 / 0) produce recombinant protein in significantly higher amounts than in the initial growth phase, so that protein titer can be measured. The exact conditions will vary depending on the cell type, the organism from which the cells are derived, and the properties and characteristics of the recombinant protein expressed.

[0274] According to the disclosure, the production bioreactor can be any volume suitable for the large-scale production of recombinant proteins. In one aspect, the volume of the production bioreactor is at least 500 liters. In other aspects, the volume of the production bioreactor is 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000, 25,000 liters or more, or any volume in between. Those of ordinary skill in the art will know and will be able to select suitable bioreactors for implementing the disclosure. The production bioreactor can be made of any material that is conducive to cell growth and vigor that does not interfere with the expression or stability of the produced polypeptide or protein.

[0275] In some aspects, during the initial growth phase, the cells are grown for a period of time sufficient to reach a viable cell density that is a given percentage of the maximum viable cell density that the cells would eventually reach if the cells were allowed to grow undisturbed. For example, the cells can be grown for a period of time sufficient to reach a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of the maximum viable cell density.

[0276] In some respects, allow the time period of cell growth restriction.For example, according to the starting concentration of cell culture, the temperature of cell growth thereunder and the intrinsic growth rate of cell, cell can grow 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 or more days in cultivation.In some cases, can allow cell growth more than one month.If the growth of cell in seed bioreactor is enough, namely the viable cell density in production bioreactor has been in the expected percentage of maximum viable cell density when it is inoculated, then cell will be under the initial growth stage temperature, in production bioreactor, grow 0 day.

[0277] The cell culture may be stirred or shaken during the initial growth phase to increase oxygenation and dispersion of nutrients to the cells. In accordance with the present disclosure, certain internal conditions of the bioreactor may be controlled or adjusted during the initial growth phase, including, but not limited to, pH, temperature, oxygenation, etc. For example, pH may be controlled by supplying appropriate amounts of acid or base, and oxygenation may be controlled using sparging devices well known in the art.

[0278] Various methods for culturing mammalian cells for the production of recombinant proteins by batch, fed-batch, continuous, semi-continuous and perfusion culture modes are known in the art. See, for example, Willard, SS, M., BioProcess Int. 15(3)38-46 (2017) for a discussion of culture methods in monoclonal antibody production.

[0279] In preferred aspects, the rat hybridoma cells as described herein are cultured using a fed-batch mode of operation. As used herein, the term "fed-batch" or "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culture process. In some aspects of the fed-batch method, nutrients are added once the nutrients are depleted. Fed-batch culture can use a basal medium (e.g. ) starts. A culture medium that provides additional components to the culture at some time after the start of the culture process is a feed medium (e.g., BalanCD CHO Feed Fed-batch cultures are usually stopped at some point (based on number of days in culture or cell viability, whichever comes first), and the cells and / or components in the culture medium are harvested and purified.

[0280] Alternative culture modes such as batch and perfusion modes of operation are also contemplated for use with the methods of the present disclosure, however, it will be appreciated by those skilled in the art that some modifications may need to be made using routine experiments. For example, it will be appreciated by those skilled in the art that in batch mode, feed media will not be used. As used herein, "batch culture" or "batch mode of operation" refers to a cell culture mode in which cells are grown in a fixed volume of nutrient medium under specific environmental conditions until a certain density is reached, and then harvested and processed as a batch before nutrients are exhausted. As used herein, "perfusion culture" or "perfusion mode of operation" refers to a cell culture mode in which a continuous flow of a physiological nutrient solution at a stable rate is passed through or flows through a cell population.

[0281] Monitoring cell culture conditions

[0282] In some aspects of the disclosed methods, specific conditions of growing cell cultures are regularly monitored. Monitoring cell culture conditions allows the practitioner to determine whether the cell culture is producing recombinant protein at suboptimal levels, or whether the culture is about to enter a suboptimal production phase. In order to monitor certain cell culture conditions, it will be necessary to remove a small aliquot of the culture for analysis.

[0283] As non-limiting examples, monitoring the temperature, pH, cell density, viable cell density, cell viability, integrated viable cell density, lactic acid level, ammonium level, osmotic pressure, amount of dissolved oxygen, pCO2 level, glutamine level, glutamate level or glucose level or titer of expressed polypeptide or protein of cell culture may be beneficial or necessary. In some aspects, such parameters are measured regularly. In some aspects, such parameters are measured more than once a day (i.e. 1, 2, 3, 4, 5). In some aspects, such parameters are measured every day. In some aspects, such parameters are measured every other day. In some aspects, such measurements are carried out during the protein production phase of cell culture. In some aspects, such measurements are carried out during the initial growth phase of cell culture. In some embodiments, the expression or activity level of the expressed recombinant protein is measured on the 1st, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after starting cell culture.

[0284] Many techniques well known in the art will allow those of ordinary skill in the art to measure these conditions. For example, cell density can be measured using a hemocytometer, a Coulter counter or a cell density check (CEDEX). The viable cell density can be determined by staining the culture sample with trypan blue. Since only dead cells absorb trypan blue, the viable cell density can be determined by counting the total number of cells, dividing the number of cells that absorb the dye by the total number of cells, and taking the reciprocal. Cell viability can also be measured using a biomass capacitance probe. HPLC can be used to determine the level of lactic acid, ammonium or expressed polypeptide or protein. Alternatively, the level of expressed polypeptide or protein can be determined by standard molecular biology techniques such as Coomassie brilliant blue staining, Western blotting, Bradford assay, Lowry assay, Biuret assay and UV absorbance of SDS-PAGE gel. Monitoring the post-translational modification (including phosphorylation and glycosylation) of the expressed polypeptide or protein can also be beneficial or necessary.

[0285] Changing process parameters in rat hybridoma (e.g. YB2 / 0) cell culture

[0286] In some aspects of the present disclosure, rat hybridoma cells are cultured under culture conditions that promote and optimize the production, titer, and product quality of the recombinant protein (e.g., monoclonal antibody) to be expressed. For example, in some aspects, the cell culture can be transformed by changing one or more of a number of culture conditions, including, for example, temperature, pH, osmotic pressure, and sodium butyrate levels.

[0287] In some aspects, the following process parameters can be shifted during the protein production phase of cell culture (culture day 3→harvest). In certain aspects, process parameters can be shifted during the initial growth phase of cell culture (culture day 0→2).

[0288] In some aspects, the process parameter transitions described below do not occur simultaneously. In some aspects, the process parameter transitions described below can occur simultaneously, or within at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 hours of each other. In some aspects, a particular process parameter transition (e.g., temperature) does not typically precede or follow another process parameter transition (e.g., pH). As disclosed herein, process parameter transitions can occur alone or in combination.

[0289] Controlling pH and pCO2 levels

[0290] Both pCO2 and culture pH affect the product quality profile of recombinant glycoproteins. Brunner, M. et al., Bioprocess and Biosystems Engineering 40:251-263 (2017). pH control strategies are critical to controlling pCO2 levels. pH is an important process control parameter. Culture pH is critical to mammalian cell physiology. The typical pH control range for mammalian cell lines in culture is 6.7-7.3. To help support pH maintenance in cell culture media, various buffers are supplemented to the cell culture media. Sodium bicarbonate is one of the most common. Alternative buffer systems including HEPES are also commonly used. See, Itagaki, A. et al., Experimental Cell Research 83:351-361 (1974). For sodium bicarbonate buffers, control of culture pH is usually two-sided. When the pH exceeds the desired value, carbon dioxide is added to the bioreactor to reduce the pH to an acceptable value. When the pH drops below the desired value, a diluted alkaline solution is usually added to the bioreactor to increase the pH to an acceptable value. Alternative strategies for pH control in mammalian cell bioreactors have been queried. The choice of pH control methodology and buffer system ultimately depends on the manufacturer of the therapeutic protein. Excessive alkali and CO2 addition and accumulation in bioreactors have been shown to be detrimental to the growth of mammalian cells in culture. See deZengotita, V. et al., Cytotechnology 28:213-227 (1998). Therefore, controlling the level of dissolved CO2 (pCO2) and alkali addition is important for overall culture process performance. Therefore, cell culture process variables such as pH and pCO2 are closely monitored during manufacturing. The same is true for the product quality of the expressed protein after harvesting and purification from cell culture.

[0291] pH

[0292] In some aspects, the method for manufacturing a recombinant protein in a rat hybridoma cell includes culturing a rat hybridoma cell in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cell includes an expression vector comprising a polynucleotide encoding the recombinant protein. In some aspects, the culture pH is about 6.5 to about 7.0. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the second day of the culture of the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is set at the third day of the culture of the cell culture. In some aspects, the culture pH is about 7.0 to about 7.55. In some aspects, the culture pH of about 7.0 to about 7.55 is set at the 0th to the third day of the culture of the cell culture.

[0293] In some aspects, the culture pH is reduced (or converted) to about 6.5 to about 7.0 on the 2nd or 3rd day of cell culture. In some aspects, the culture pH is reduced on the 3rd day of cell culture. In some aspects, from the 3rd day of culture of cell culture until harvest, a culture pH of about 6.5 to about 7.0 is maintained. In some aspects, the timing of the pH conversion on day 3 occurs in a production bioreactor 62-77 hours after inoculation.

[0294] In some aspects, the harvest titer of the recombinant protein is increased and / or the fucosylation of the recombinant protein is reduced when the culture pH is 6.6 to 6.96 relative to cell culture under the same culture conditions except that the culture pH is 6.60 to 6.8.

[0295] Another pH-related process parameter monitored in the rat hybridoma cell culture disclosed herein is called "integrated pH2 difference". See Example 5. The integrated pH2 difference refers to the cumulative culture time and cumulative decline in pH allowed to drop below the fixed pH set point after the pH is reduced on the third day of culture. The integrated pH2 difference increases when the culture pH drops below the fixed pH set point for any time measurement unit. The fixed pH set point is a specified value. In some aspects, the fixed pH set point is pH 6.91. In some aspects, relative to cell cultures with a large integrated pH2 difference, the integrated pH2 difference of cell cultures involving a fixed pH set point (e.g., pH 6.91) is smaller. In some aspects, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and a higher titer at harvest. In some aspects, a lower integrated pH2 difference further results in a lower fucosylation percentage.

[0296] pCO2

[0297] In a cell culture medium containing a sodium bicarbonate-based buffer, CO2 is introduced into the cell culture to control pH. When the pH increases above the target value / range, CO2 is added to reduce the pH. The exogenously added CO2 dissolves in the culture medium, increasing the pCO2 level. In some aspects, as described herein, the culture conditions further include controlling the culture pCO2 level to be less than about 300 mmHg by virtue of the control of the culture pH. As used herein, "less than about 300 mmHg" means that the pCO2 level can range from 0 to 300 mmHg.

[0298] In addition to higher pH set points, there are a variety of cell culture process means to support higher pH control levels while controlling low pCO levels. Alternative means to promote low pCO levels include increasing the buffering capacity of the cell culture medium by supplementing additional buffers such as HEPES. This will weaken the cultivation-related increase of pCO during the culture period by reducing the need to add CO2 to the bioreactor to control pH. Another alternative means to promote low pCO levels in the bioreactor is to increase air overlay and air injection rate to increase the pCO2 stripping (stripping) capacity of the bioreactor and prevent high levels from being achieved. Increasing the dissolved oxygen (DO) set point and / or reducing the bioreactor agitation rate will have a similar effect of forcing higher air and O2 flows into the bioreactor to maintain DO, effectively flushing excess pCO2 out of the bioreactor.

[0299] In some aspects of the methods of the present disclosure, pCO2 levels of less than about 300 mmHg are promoted by supplementing the cell culture with additional buffer, increasing the air sparging rate, increasing the DO set point, and / or reducing the agitation rate.

[0300] In some aspects, the cell culture is carried out in a commercial scale bioreactor. In some aspects, the commercial scale bioreactor is a 10,000L, 15,000L, 20,000L or 25,000L bioreactor. In some aspects, the commercial scale bioreactor is a 15,000L bioreactor.

[0301] In some aspects, the rat hybridoma cell is a YB2 / 0 rat hybridoma cell.

[0302] In some aspects, the recombinant protein is an IgG1 glycoprotein.

[0303] In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0304] In some aspects, the monoclonal antibody specifically binds an epitope of CD20 (ie, is an anti-CD20 antibody).

[0305] In some aspects, the monoclonal antibody is TG- 1101. In some aspects, the monoclonal antibody binds to the same epitope as TG-1101.

[0306] In some aspects, additional cell culture process parameters such as, for example, temperature control, as described below, are shifted in conjunction with shifts in culture pH or alternative shifts in pCO2 to optimally express a recombinant protein of interest (e.g., a monoclonal antibody) in rat hybridoma cells.

[0307] Temperature transition

[0308] In the methods of the present disclosure, the temperature shift of cell culture is another culture condition for increasing expression, cell density or viability, and product quality of recombinant proteins in rat hybridoma cell lines. In some aspects, it may also be desirable to use multiple discrete temperature shifts at different times during cell culture.

[0309] In some aspects, the temperature shift of the cell culture of the rat hybridoma cells is combined with a pH shift as described herein. In certain aspects, the temperature shift of the cell culture is combined with a pH shift and is combined with the use of a chemically defined cell culture medium for ADCF. In some aspects, the chemically defined cell culture medium for ADCF is In some aspects, the feed medium BalanCD CHO Feed and Use together.

[0310] In some aspects, the culture conditions include an initial temperature set point of about 37°C, wherein the initial temperature set point is set between culture day 0 and culture day 1.

[0311] In some aspects, the culture conditions further include a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3. As used herein, "the end of culture day 1" refers to 17 to 33 hours after starting cell culture.

[0312] In some aspects, the culture conditions further include a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest. In some aspects, the third temperature set point is 32.5°C.

[0313] In some aspects, the cell culture comprises the following culture conditions: i). an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set at culture day 3 and maintained until harvest; ii). a culture pH between about 6.5 and about 7.55; and iii). a culture pCO2 of less than about 300 mmHg.

[0314] In some aspects, "culture day 1" refers to about 12.0 hours to about 35.9 hours after the cells in the seed culture are inoculated into the production bioreactor. In some aspects, "culture day 2" refers to about 36.0 hours to about 59.9 hours after the cells in the seed culture are inoculated into the production bioreactor. In some aspects, "culture day 3" refers to about 60.0 hours to about 83.9 hours after the cells in the seed culture are inoculated into the production bioreactor.

[0315] In some aspects, the timing of the first temperature shift occurs in the production bioreactor 17-33 hours after inoculation. In some aspects, the timing of the second temperature shift occurs in the production bioreactor 62-77 hours after inoculation.

[0316] In some aspects, as described herein, when the culture pH is controlled and the temperature is shifted, the recombinant protein harvest titer in the rat hybridoma cells is about 0.5 g / L to about 1.5 g / L. In some aspects, the recombinant protein harvest titer in the rat hybridoma cells is about 0.5 g / L to about 1.3 g / L. In some aspects, the recombinant protein harvest titer in the rat hybridoma cells is about 1.0 g / L to about 1.5 g / L.

[0317] In some aspects, the yield of the recombinant protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%, relative to a recombinant protein produced by a culture process that does not employ: i) an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0318] In some aspects, as described herein, when the culture pH is controlled and the temperature is shifted, the fucosylation percentage of the prepared recombinant protein is between about 20% and about 35%. In some aspects, as described herein, when the culture pH is controlled and the temperature is shifted, the fucosylation percentage of the prepared recombinant protein is about 20% to about 30%.

[0319] In some aspects, the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein comprises between 20% and 40% fucosylated glycans; between 23% and 36% fucosylated glycans; between 28% and 33% fucosylated glycans; or about 33% or about 36% fucosylated glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Fucosylated glycans are polysaccharides that carry fucose residues ( Figure 1 (shown as a hollow triangle) Figure 1 In brief, a sample or population of anti-CD20 antibodies undergoes enzymatic deglycosylation so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans in the N-glycans cleaved using enzymatic digestion.

[0320] In certain aspects, the recombinant protein or anti-CD20 antibody made by the methods disclosed herein comprises at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or at least 40%, and at most 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or at most 40% fucosylated glycans. In some aspects, the recombinant protein or anti-CD20 antibody comprises about 36% fucosylated glycans.

[0321] In some aspects, when the culture pH is controlled and the temperature is shifted, as described herein, the sialylation of the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein is about 1% to about 4%.

[0322] In some aspects, the recombinant protein or anti-CD20 antibody made by the methods disclosed herein comprises less than 10%, 8%, 5%, 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some aspects, the recombinant protein or anti-CD20 antibody made by the methods disclosed herein comprises between 10% and 0.5% sialylated glycans; between 10% and 5% sialylated glycans; between 5% and 0.5% sialylated glycans; between 4% and 0.5% sialylated glycans; between 2% and 0.5% sialylated glycans; or undetectable amounts of sialylated glycans. In short, a sample or population of anti-CD20 antibodies undergoes enzymatic deglycosylation so that all N-glycans are cut from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of sialylated N-glycans is the percentage of sialylated N-glycans among N-glycans cleaved using enzymatic digestion.

[0323] In certain aspects, the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein comprises at least undetectable amounts, 0.5%, 1%, 2%, 3%, 4%, or at least 5% and at most 0.5%, 1%, 2%, 3%, 4%, 5%, or at most 10% sialylated glycans. In some aspects, the recombinant protein or anti-CD20 antibody comprises undetectable amounts of sialylated glycans.

[0324] In some aspects, the temperature-shifted and pH-controlled culture conditions as described herein further include culture in a chemically defined, animal-derived component-free (ADCF) cell culture basal medium such as, for example, (Cytiva Lifesciences / GE) and feed medium BalanCD CHO Feed (Irvine Scientific). In some aspects, feed medium is added to the cell culture approximately every 48 hours. In some aspects, glucose, glutamine and / or cholesterol lipid solution are added to supplement the feed medium in the cell culture.

[0325] In some aspects, the methods for producing recombinant proteins in rat hybridoma cells as described herein produce a recombinant protein harvest titer of about .5 g / L to about 1.3 g / L, the methods utilizing cell culture pH control and temperature control, and in a chemically defined and animal derived component free (ADCF) cell culture medium, e.g. and feed medium BalanCD CHO Feed In some aspects, the cells are cultured in a bioreactor. In some aspects, the cells are cultured in a commercial scale bioreactor (e.g., 10,000 L to 25,000 L). In some aspects, the cells are cultured in a 15,000 L bioreactor.

[0326] Cell culture media and feed media

[0327] Hundreds of basal cell culture media formulations for mammalian cell culture are well known in the art and commercially available, including serum-free, peptone-free, animal-derived component-free (ADCF) and / or chemically defined media. To these basal media formulations, the skilled artisan will add components such as, for example, amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements, etc., according to the requirements of the specific type of host cells to be cultured.

[0328] Cell culture media typically include at least one or more components from the following: an energy source (e.g., in the form of carbohydrates such as glucose); essential amino acids, including the twenty basic amino acids plus cysteine; vitamins and / or other organic compounds that are usually required at low concentrations; lipids or free fatty acids (e.g., linoleic acid); and trace elements (e.g., inorganic compounds or naturally occurring elements that are usually required at very low concentrations, often in the micromolar range). The culture medium can be solid, gelatinous, liquid, gaseous, or a mixture of phases and materials.

[0329] Some examples of commercially available cell culture basal media are listed in Table 2 of Example 1, and include, for example, Hycell Medium, IS CHO-SD G10.6 Medium, CD Hybridoma, PFHM-II Protein Free Hybridoma, Ex-Cell CD Hybridoma, UltraDOMA-PF Hybridoma, ProDOMA 1, ProDOMA 1, Liquid Medium (LM) | CDM4Mab, and ActiCHO-P.

[0330] In addition, the cell culture medium may be optionally supplemented to include one or more additional components at appropriate concentrations or amounts, as needed or desired, and as will be known and practiced by those of ordinary skill in the art. Supplements that support the growth and maintenance of a particular cell culture can be readily determined by those of ordinary skill in the art, such as described in, for example, Barnes et al., Cell 22:649 (1980); Mammalian Cell Culture, Mather, JP, ed., Plenum Press, NY (1984); and US Patent No. 5,721,121.

[0331] Exemplary supplements include, but are not limited to, chemical gene selectors, hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, somatotropin, pituitary extract, aprotinin); salts (e.g., calcium, magnesium and phosphate), and buffers (e.g., HEPES (4-[2-hydroxyethyl]-1-piperazine-ethanesulfonic acid)); nucleosides and bases (e.g., adenosine, thymidine, hypoxanthine); proteins and hydrolysates; antibiotics (e.g., gentamicin); cytoprotectants (e.g., Pluronic polyols (PLURONIC.RTM.F68)) and extracellular matrix proteins (e.g., fibronectin).

[0332] In some aspects, a culture medium suitable for the method of the present disclosure is supplemented with a feed medium. In some aspects, the feed medium is a feed medium with a clear chemical composition. In some aspects, a feed medium (media) (or CDFM) or a culture medium (medium) with a clear chemical composition refers to a culture medium containing more than one nutrient whose chemical composition and relative concentration are known, and is added to the culture medium at a certain time after inoculation. With or without regular cell and / or product harvesting before the end of cell culture, CDFM is continuously or in discrete increments supplied to the culture container and supplied to the culture medium during culture. CDFM can be individually formulated as a unique mixture of amino acids, vitamins, trace minerals and organic compounds contained in an enriched amount as a feed medium for cell culture medium. Alternatively, commercially available CDFM can be used. Some examples of commercially available CDFM are listed in Table 3 of Example 1, and include EX-CELL Advanced CHO Feed 1 (containing glucose); Cell Boost 6; CHO CD Efficient Feed A; BalanCD CHO feed 4; Cell Boost 7A and b; and Cell Boost 3.

[0333] In some aspects, the basal medium (Cytiva Lifesciences / GE) and feed medium BalanCD CHO Feed (Irvine Scientific) combination for rat hybridoma cell culture.

[0334] As disclosed in Example 1, a number of cell culture medium formulations and feed media were investigated to maximize cell growth, cell viability, productivity, fucosylation percentage and Fc effector function of recombinant antibodies derived from rat hybridoma cell lines (e.g., YB2 / 0 cells) in culture.

[0335] In certain aspects, the disclosure relates to methods of producing a recombinant protein in a rat hybridoma cell line, the method comprising culturing the cells in cell culture, wherein the rat hybridoma cells are cultured in a basal medium and a feed medium suitable for rat hybridoma cells.

[0336] In some aspects, rat hybridoma cells are cultured in chemically defined animal component-free (ADCF) medium.

[0337] In some aspects, the rat hybridoma cells are (Cytiva) and feed medium BalanCDCHO Feed (Irvine Scientific). In some aspects, the basal medium and feed medium BalanCD CHO Feed In some aspects, other basal media (except In some aspects, other feed media (except BalanCD CHO Feed For the rat hybridoma cell culture described herein.

[0338] In some aspects, the basal medium, e.g. In some aspects, BalanCD CHO Feed Add on the 3rd day of cell culture.

[0339] In some aspects, feed medium such as BalanCD CHO Feed Feed medium is added to the cell culture approximately every 48 to 72 hours. In some aspects, the feed medium, such as BalanCD CHO Feed Feed medium is added to the cell culture approximately every 48 hours. Feed medium is added to the cell culture approximately every 72 hours. In some aspects, glucose, glutamine and / or cholesterol lipid solution are added to supplement the feed medium BalanCD CHO Feed in the cell culture.

[0340] Compared with the basal medium and feed medium BalanCD CHO Feed Cells cultured in basal medium and feed medium BalanCD CHO Feed In some aspects, the recombinant protein cultured in the basal medium is produced at an increased harvest titer. and feed medium BalanCD CHO Feed The harvest titer of the recombinant protein cultured in the basal medium is about 0.3 g / L to about 1.5 g / L. In some aspects, and feed medium BalanCD CHO Feed The harvest titer of the recombinant protein cultured in the basal medium is about 0.3 g / L to about 1.3 g / L. In some aspects, and feed medium BalanCD CHO Feed The harvest titer of the recombinant protein cultured in is about 0.5 g / L to about 1.0 g / L. In some aspects, the harvest titer of the recombinant protein is about 1.0 g / L.

[0341] In some aspects, relative to not in the basal medium and feed medium BalanCD CHOFeed Cells cultured in basal medium and feed medium BalanCD CHO Feed In some aspects, the percentage of fucosylation of the recombinant protein cultured in the basal medium is reduced. and feed medium BalanCD CHO Feed The percentage of fucosylation of the recombinant protein cultured in the basal medium is reduced to about 18% to about 73%. In some aspects, the fucosylation of the recombinant protein cultured in the basal medium is reduced to about 18% to about 73%. and feed medium BalanCD CHO Feed The percentage of fucosylation of the recombinant protein cultured in the basal medium is reduced to about 18% to about 40%. In some aspects, and feed medium BalanCD CHO Feed In some aspects, the fucosylation percentage of the recombinant protein cultured in is reduced to about 18% to about 30%. In some aspects, the fucosylation percentage of the recombinant protein is reduced to about 18%.

[0342] In certain aspects, in the basal medium and feed medium BalanCD CHO Feed The rat hybridoma cell line cultured in is the YB2 / 0 rat hybridoma cell line.

[0343] In some aspects, in the basal medium and feed medium BalanCD CHO Feed The recombinant protein produced in the rat hybridoma cell line cultured in basal medium is an IgG1 glycoprotein. and feed medium BalanCD CHO Feed The recombinant protein produced in the rat hybridoma cell line cultured in is a monoclonal antibody. In some aspects, the monoclonal antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0344] In some aspects, in the basal medium and feed medium BalanCD CHO Feed The monoclonal antibody produced in the rat hybridoma cell line cultured in basal medium specifically binds to the epitope of CD20. In some aspects, the monoclonal antibody produced in the rat hybridoma cell line cultured in basal medium specifically binds to the epitope of CD20. and feed medium BalanCD CHO Feed The monoclonal antibody produced in the rat hybridoma cell line cultured in vitro is the anti-CD20 antibody TG-1101.

[0345] In some aspects, relative to not in the basal medium and feed medium BalanCD CHOFeed The percentage of FcγRIIIa binding of monoclonal antibodies cultured in basal medium and feed medium BalanCD CHO Feed In some aspects, the FcγRIIIa-158V binding percentage of the monoclonal antibody produced in the rat hybridoma cell line cultured in the culture medium increases. In some aspects, the FcγRIIIa-158V binding percentage increases by about 5% to about 30%. In some aspects, the FcγRIIIa-158V binding percentage increases by about 20% to about 30%. In some aspects, the FcγRIIIa-158V binding percentage increases by about 20%. In some aspects, the FcγRIIIa-158V binding percentage is evaluated by surface plasmon resonance (SPR).

[0346] In some aspects, relative to not in the basal medium and feed medium BalanCD CHOFeed The percentage of ADCC activity of monoclonal antibodies cultured in basal medium and feed medium BalanCD CHO Feed In some aspects, the percentage of antibody dependent cellular cytotoxicity (ADCC) activity of the monoclonal antibody produced in the rat hybridoma cell line cultured in vitro increases. In some aspects, the ADCC activity percentage increases by about 10% to about 70%. In some aspects, the ADCC activity percentage increases by about 50% to about 70%. In some aspects, the ADCC activity percentage increases by about 60% to about 70%. In some aspects, the ADCC activity percentage is evaluated by a cell-based bioassay.

[0347] In some aspects, in the basal medium and feed medium BalanCD CHO Feed The cell culture is carried out in a bioreactor. In some aspects, the bioreactor is a commercial scale bioreactor (eg, 15,000 L, 20,000 L, or 25,000 L volume).

[0348] In some aspects, other cell culture process parameters such as, for example, pH and temperature control as described herein are combined with the basal medium. and feed medium BalanCD CHO Feed The use of a combination of transformants is used to optimally express recombinant proteins (eg, antibodies) in rat hybridoma cell lines (eg, YB2 / 0).

[0349] Harvesting recombinant proteins and assessing protein quality, titer, and potency

[0350] In some aspects, the method of making a recombinant protein further comprises harvesting the recombinant protein produced by the rat hybridoma cells. In some aspects, harvesting of the cell culture will occur on culture day 12 or 13 of the cell culture, or when cell viability drops below 20%, whichever comes first.

[0351] In general, it will generally be desirable to isolate and / or purify the proteins or antibodies expressed according to the present disclosure. In preferred aspects, the expressed polypeptide or protein is secreted into the culture medium, and thus cells and other solids may be removed as a first step in a purification process, such as by centrifugation or filtration.

[0352] Alternatively, the expressed polypeptide or protein is bound to the surface of the host cell. In this embodiment, the culture medium is removed and the host cells expressing the polypeptide or protein are lysed as the first step in the purification process. The lysis of mammalian host cells can be achieved by many means well known to those of ordinary skill in the art, including physical disruption by glass beads, exposure to high pH conditions, exposure to freezing temperatures, and the addition of a cell lysis buffer containing a detergent.

[0353] The polypeptide or protein can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, size exclusion chromatography, and hydroxyapatite chromatography), gel filtration, centrifugation or differential solubility, ethanol precipitation, or by any other available technique for purification of proteins (see, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all of which are incorporated herein by reference). In particular, for immunoaffinity chromatography, protein can be separated by being bound to an affinity column comprising an antibody, which is produced for the protein and attached to a fixed support. Alternatively, affinity tags such as influenza capsid sequences, polyhistidine or glutathione-S-transferases can be attached to proteins by standard recombinant techniques to allow easy purification by passing through (passage over) an appropriate affinity column. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin can be added at any or all stages to reduce or eliminate the degradation of polypeptide or protein during the purification process. When cells must be lysed in order to separate and purify expressed polypeptides or proteins, protease inhibitors are particularly desirable. It will be appreciated by those of ordinary skill in the art that definite purification techniques will vary according to the composition of the culture medium in which the polypeptide or protein to be purified, the characteristics of the cells from which the polypeptide or protein is expressed, and the cells grow therein.

[0354] In some aspects, the methods of the present disclosure further comprise purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some aspects, the affinity chromatography comprises protein A purification. In some aspects, the purified recombinant protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation.

[0355] In some aspects, after the expressed recombinant protein is harvested and purified from the cell culture, the product quality is evaluated.

[0356] In some aspects, the quality of the purified recombinant protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis. In some aspects, the purified recombinant protein (e.g., monoclonal antibody) is measured for percent fucosylation by performing N-linked glycan analysis, as described, e.g., in Examples 1, 2, and 6.

[0357] In some aspects, the purified recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody specifically binds to an epitope of CD20 (also referred to herein as an "anti-CD20 antibody"). In some aspects, the anti-CD20 antibody specifically binds to the same epitope as TG-1101.

[0358] In some aspects, the biological properties of the anti-CD20 antibodies manufactured by the methods disclosed herein can be measured and described in the assay by using a comparison with a reference standard. In some aspects, the reference standard is a commercial reference standard. In some aspects, the commercial reference standard is RS-117808. RS-117808 ("antibody urituximab (TG-1101)") is deposited in the American Type Culture Collection (ATCC) according to the terms of the Budapest Treaty, located at 10801 University Boulevard, Manassas, VA 20110, received by ATCC on April 15, 2022, and assigned informal patent deposit number PTA-127294.

[0359] In some aspects, the reference standard is an anti-CD20 antibody. In certain aspects, the reference standard is GAZYVA (otuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), velutuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab).

[0360] In some aspects, the monoclonal antibodies are subjected to more than one binding assay to evaluate CD20 binding activity (or other antibody binding if the antibody is not an anti-CD20 antibody), FcγRIIIa-158V binding and / or C1q binding. In some aspects, the recombinant antibodies are assayed for antibody-dependent cellular cytotoxicity (ADCC) bioactivity / potency and / or complement-dependent cytotoxicity (CDC) bioactivity / potency, such as described in Examples 1 and 2. Additional assays to assess protein or antibody quality or function are well known to those of skill in the art.

[0361] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 82% to 138%% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 92% to 118%% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 109% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some aspects, the percentage of CD20 binding is determined by binding of an anti-CD20 antibody to a human mantle cell lymphoma cell line Jeko-1 expressing CD20, based on a cell-based CD20 binding activity bioassay.

[0362] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 82% to 130% FcγRIIIa-158V binding relative to a commercial reference standard as measured by surface plasmon resonance (SPR). In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 76% to 130% FcγRIIIa-158V binding relative to a commercial reference standard as measured by surface plasmon resonance (SPR).

[0363] In some aspects, as measured by surface plasmon resonance, the monoclonal antibody has a KD value of 30 to 70 nM in the FcγRIIIa-158V binding assay. In some aspects, as measured by surface plasmon resonance, the monoclonal antibody has a KD value of about 59 nM in the FcγRIIIa-158V binding assay. In some aspects, as measured by surface plasmon resonance, the monoclonal antibody has a KD value of 500 to 1000 nM in the FcγRIIIa 158F binding assay. In some aspects, as measured by surface plasmon resonance, the monoclonal antibody has a KD value of 760 nM in the FcγRIIIa 158F binding assay. In some aspects, compared with the anti-CD20 antibody rituximab, the monoclonal antibody has a significantly higher binding affinity to FcγRIIIa 158V or FcγRIIIa 158F.

[0364] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 86 to 117% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 86 to 116% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of 88 to 113% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative potency of about 99% in a C1q binding assay as measured by ELISA compared to a commercial reference standard.

[0365] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity relative to monoclonal antibodies produced by a culture process that does not employ the following culture conditions: i) an initial temperature set point of about 37°C, a second temperature set point of about 35°C, and a third temperature set point of about 32.5°C; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0366] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure induce greater cytotoxicity in a cell-based antibody-dependent cellular cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0367] In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative efficacy of 90% to 163% in cell-based ADCC assays compared to commercial reference standards. In some aspects, the monoclonal antibodies produced by the methods of the present disclosure have a relative efficacy of about 117% in cell-based ADCC assays compared to commercial reference standards. In some aspects, cell-based ADCC assays use effector cells selected from CD16 effector cells and primary NK cells. In some aspects, the group performs more than 100% of the commercial reference standards in cell-based ADCC using CD16 effector cells.

[0368] In some aspects, compared with commercial reference standards, the monoclonal antibodies produced by the method of the present disclosure have a relative efficacy of 74% to 127% in cell-based complement dependent cytotoxicity (CDC) assays. In some aspects, compared with commercial reference standards, the monoclonal antibodies produced by the method of the present disclosure have a relative efficacy of 73% to 128% in cell-based complement dependent cytotoxicity (CDC) assays. In some aspects, compared with commercial reference standards, the monoclonal antibodies produced by the method of the present disclosure have a relative efficacy of 78 to 116% in cell-based CDC assays. In some aspects, compared with commercial reference standards, the monoclonal antibodies produced by the method of the present disclosure have a relative efficacy of about 91% in cell-based CDC assays.

[0369] In some aspects, the monoclonal antibody produced by any of the methods disclosed herein comprises: a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0370] In some aspects, the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7 and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0371] In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:9. In some aspects, the monoclonal antibody comprises a deletion of up to 5 N-terminal residues. In some aspects, the monoclonal antibody comprises a deletion of up to 10 N-terminal sequences.

[0372] In some aspects, the methods of the present disclosure produce a harvest titer of a recombinant protein (e.g., anti-CD20 antibody) of about .5 g / L to about 1.5 g / L. In some aspects, the harvest titer is about 1.0 g / L to about 1.5 g / L.

[0373] In some aspects, the anti-CD20 antibody is TG-1101 or an antibody that binds to the same epitope as TG-1101. In some aspects, the anti-CD20 antibody is TG-1101.

[0374] Commercial-scale production of recombinant proteins in rat hybridoma cells

[0375] Commercial scale (e.g., 10,000 L-25,000 L) production of recombinant proteins (e.g., anti-CD20 antibodies) expressed in rat hybridoma cells is also provided. See Example 3.

[0376] In some aspects, a method for producing a recombinant protein in culture of rat hybridoma cells at a commercial scale is provided, the method comprising the steps of: a) preparing and thawing a working rat hybridoma cell bank of a recombinant protein of interest; b) expanding the size and volume of the culture of rat hybridoma cells from the cell bank through a series of shake flasks (125 mL, 500 mL, 3 L, 3x3 L shake flasks, and 50 L cell bags) with a target seeding density of at least 0.30 x 10 6 viable cells / mL; c) process the cell culture through a series of seed bioreactors (120L, 600L, and 3,000L) to further increase the volume and cell culture quality; d) inoculate the cell culture from the 3,000L seed bioreactor into the 15,000L production bioreactor; e) harvest the cell culture supernatant from the production bioreactor; f) clarify the recovered cells by serial centrifugation followed by depth filtration; g) purify the recombinant protein by protein A capture column chromatography; and h) inactivate viral material by solvent detergent viral inactivation (SDVI).

[0377] In some aspects, protein A column chromatography is performed to purify the recovered recombinant protein and reduce process impurities such as cell culture components, HCPs and residual DNA, as well as to provide viral safety. In some aspects, protein A column chromatography is performed using MabSuReSelect resin (Cytiva) in bind / elute mode.

[0378] In some respects, the HETP performance of packed column is assessed using sodium acetate / benzyl alcohol buffer. In some respects, post operation is carried out at 13 to 25 ℃. In some respects, before loading, post is sterilized with 0.5M sodium hydroxide, rinsed with WFI, and balanced with equilibrium buffer (25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1). In some respects, the harvest through clarification is mixed for a short time, then loaded onto the post using a maximum of 21g recombinant protein / L resin load, and the post is cleaned with Wash1Buffer (equilibrium buffer), then cleaned for the second time with high salt Wash 2Buffer (25mM Tris, 1.2M NaCl, 5mMEDTA, pH 7.1), then additional cleaning is carried out using Wash 3Buffer (equilibrium buffer). In some respects, the harvest through clarification is loaded onto the post using a maximum of 36g recombinant protein / L resin load. The elution peak collection was used by A280, and the recombinant protein bound was eluted with elution buffer (25mM sodium citrate, pH 3.6) at 200-220cm / h, no more than 1.2 times of column volume. In some aspects, the elution peak collection was used by A280, and the recombinant protein bound was eluted with elution buffer (25mM sodium citrate, pH3.6) at 200-220cm / h, no more than 2.4 times of column volume. The eluate was collected in a tank containing neutralization buffer (2.0M Tris, pH 7.5), and filtered via a 0.2 μm filter before being transferred to different tanks. Protein A columns were disinfected with 0.5M sodium hydroxide. Three cycles can be run at most for each batch; if the protein A process requires multiple cycles, the column is rebalanced with equilibrium buffer to carry out the next cycle. After disinfection, the protein A column is neutralized with equilibrium buffer, and stored at 13 to 25°C in 200mM sodium acetate, 2% benzyl alcohol, pH 5.0. The combined (if more than one cycle), neutralized eluate is diluted with 5 mM sodium phosphate, pH 7.2, to a concentration of ≤10 g / L and stored at 13-25° C. for ≤72 hours or at 2 to 8° C. for ≤11 days. In some aspects, the combined (if more than one cycle), neutralized eluate is diluted with 5 mM sodium phosphate, pH 7.2, to a concentration of ≤10 g / L and stored at 13-25° C. for ≤24 hours.

[0379] In some aspects, the protein A capture chromatography step is followed by a solvent detergent virus inactivation (SDVI) step to inactivate potential viral material. In some aspects, the protein A elution pool is diluted and treated with 3.5% (v / v) TnBP, 12% (w / v) polysorbate 80, and kept at least 120 minutes at 24.0-26.0°C while mixing. The SDVI pool is filtered with a 0.2 μm filter before being transferred to different tanks, diluted to 50 mOsm / kg with 5 mM sodium phosphate (pH 7.2) in the tank, and pH is adjusted to 7.2 as needed. After pH adjustment, the pool is maintained at 13 to 25°C for ≤30 hours, then enters a CEX column. In some aspects, the pool is kept at 13 to 25°C for ≤24 hours.

[0380] In some aspects, the method further comprises purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX).

[0381] In some aspects, the further purification of recombinant protein and the removal of residual process impurities are carried out using cation exchange column chromatography (CEX). In some aspects, CEX is carried out using SP Sepharose Fast Flow (Cytiva) in combination / elution mode. In some aspects, the HETP performance of the packed column is evaluated using a buffer containing sodium acetate / benzyl alcohol, and all column operations are carried out at 13 to 25 ° C. In some aspects, the post is disinfected with 0.5M sodium hydroxide, rinsed with WFI, and balanced with a balance buffer (20mM sodium phosphate, pH 7.2). Virus inactivation / diluted solution is loaded onto the post with a maximum of 65g / L resin load. The post is cleaned with Wash 1Buffer (balance buffer), and then cleaned for the second time with Wash 2Buffer (balance buffer in reverse). The recombinant protein bound is eluted using 20mM sodium phosphate, 150mM NaCl, pH 7.2, and the elution peak is collected by A280 monitoring. In some aspects, the eluent is filtered (0.2 μm) and stored at 13 to 25° C. for ≤72 hours or stored at 2 to 8° C. for ≤11 days. In some aspects, the eluent is stored at 13 to 25° C. for ≤24 hours. After elution, the post is stripped with 2M NaCl and then sterilized with 0.5M sodium hydroxide. Allow one cycle per batch. After completion, the post is sterilized (0.5M sodium hydroxide) and stored in storage buffer (200mM sodium acetate, 2% benzyl alcohol, pH 5.0).

[0382] In some aspects, the further purification of recombinant protein is carried out using anion exchange membrane chromatography. In some aspects, AEX is carried out using Mustang Q (Pall Corporation) membrane absorber (MA) filter in flow-through mode. In some aspects, membrane is disposable (i.e. each individual membrane cannot be reused), and each batch can use multiple membrane capsules (membrane capsules) under appropriate load levels. In some aspects, the eluent from the CEX step is diluted with 20mM sodium phosphate (pH 8.0), and then the pH is adjusted to 8.0. Determine the concentration and calculate the number of cycles based on protein concentration so that the load is 200 to 700g recombinant protein / L membrane load. The membrane is balanced with a balance buffer (20mM sodium phosphate, 75mM NaCl, pH 8.0), disinfected with 0.5M sodium hydroxide, rinsed with 2M NaCl, and then rinsed with WFI to prepare for loading. After loading, the membrane is rinsed with 20mM sodium phosphate, 75mM NaCl, pH 8.0 to maximize the recovery rate. The product containing effluent collected in all cycles was filtered (0.5 / 0.2 μm), diluted to ≤6 g / L with 75 mM sodium citrate, 312 mM NaCl, pH 6.0, and the pH was adjusted to 6.8. In some aspects, the conditioned AEX pool is stored at 13 to 25° C. for ≤72 hours or at 2 to 8° C. for ≤11 days. In some aspects, the conditioned AEX pool is stored at 13 to 25° C. for ≤24 hours.

[0383] In some aspects, the method further includes performing virus filtration (VF) to remove potential viruses, including small viruses such as parvovirus. In some aspects, VF is performed by filtering a Viresolve pre-filter in series with a Viresolve Pro virus filter (Millipore Sigma) at an operating temperature target range of 13-25°C. The process uses enough filters to meet sample loading restrictions. In order to filter, the filter is set in series and rinsed with WFI, tested for integrity, and then disinfected with 0.5M sodium hydroxide. Then it is rinsed with equilibrium buffer (25mM sodium citrate, 154mM NaCl, pH 6.5). The filtered Mustang Q membrane effluent is processed by a virus reduction filter.

[0384] Determine protein concentration and use to confirm membrane loading ≤ 600 g / m 2 After loading, the membrane is rinsed with equilibration buffer and subjected to a post-use integrity test. In some aspects, the viral filtrate is stored at 13 to 25°C for ≤72 hours or at 2 to 8°C for ≤11 days. In some aspects, the viral filtrate is stored at 13 to 25°C for ≤33 hours.

[0385] In some aspects, commercial scale production is in a 10,000 L to 25,000 L production bioreactor. In some aspects, commercial scale production is in a 15,000 L production bioreactor. In some aspects, commercial scale production is operated in fed-batch mode.

[0386] In some aspects, a drilled (10) gas sparger with a 4.0 mm orifice diameter is used in a 15,000 L production bioreactor.

[0387] In some aspects, the method further comprises ultrafiltration / diafiltration (UFDF).

[0388] In some aspects, the method further comprises preparing a bulk drug substance formulation comprising the recombinant protein, comprising adding polysorbate 80 to the formulation buffer to prepare the bulk drug substance formulation. In some aspects, the method further comprises subjecting the bulk drug substance formulation to 0.2 μm filtration.

[0389] In some aspects, the method further comprises filling the 6 L bag to a target fill volume of 5.50 L of the bulk drug substance formulation, and storing the bulk drug substance formulation at ≤ -35 ​​°C.

[0390] In some aspects, a recombinant protein in a bulk drug substance formulation is formulated into a pharmaceutically acceptable formulation.

[0391] In some aspects, the method further comprises testing the unprocessed bulk harvest from the 15,000-L production bioreactor for microbial and viral extraneous matter, and removing the microbial and viral extraneous matter from the 15,000-L production bioreactor.

[0392] In some aspects, the rat hybridoma cell is a YB2 / 0 cell.

[0393] In some aspects, the recombinant protein produced by the manufacturing method is a monoclonal antibody. In some aspects, the monoclonal antibody binds to the epitope of CD20 (i.e., it is an anti-CD20 antibody). In some aspects, the monoclonal antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0394] In some aspects, the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:8.

[0395] In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:8.

[0396] In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:9.

[0397] In some aspects, the monoclonal antibody is TG-1101 or an antibody that binds to the same epitope as TG-1101. In some aspects, the monoclonal antibody is TG-1101. As described below, in some aspects, the monoclonal antibodies produced by the methods disclosed herein contain a unique glycosylation signature.

[0398] Rat hybridoma master cell bank and working cell bank

[0399] Also provided herein are rat hybridoma master cell bank (MCB) compositions and rat hybridoma working cell bank (WCB) compositions, which can be used to make recombinant proteins (e.g., monoclonal antibodies) disclosed herein. See Example 3. Based on, for example, cell growth, harvest titer, productivity, and product quality, the MCB and WCB as disclosed herein are phenotypically stable for at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, and at least 75 cell generations. In some aspects, based on, for example, cell growth, harvest titer, productivity, and product quality, the MCB and WCB as disclosed herein are phenotypically stable for up to 71 cell generations. Ibid.

[0400] In some aspects, the rat hybridoma MCB provided herein comprises recombinant proteins having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all of the following parameters: i) about 11 to about 13 x 10 6cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 3% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoforms as detected by iCIEF; and / or x) about 20% to about 36% basic isoforms as detected by iCIEF.

[0401] In some aspects, the rat hybridoma MCB provided herein comprises recombinant proteins having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all of the following parameters: i) about 11 to about 13 x 10 6 cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 3% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoforms as detected by iCIEF; and / or x) about 20% to about 36% basic isoforms as detected by iCIEF.

[0402] In some aspects, the rat hybridoma WCB provided herein comprises recombinant proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 2% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoforms as detected by iCIEF; and / or x) about 14% to about 38% basic isoforms as detected by iCIEF.

[0403] In some aspects, the rat hybridoma WCB provided herein comprises recombinant proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomers as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimers as detected by SEC; vi) aggregates at undetectable levels to about 2% levels as detected by SEC; vii) fragments at undetectable levels to about 1% levels as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoforms as detected by iCIEF; and / or x) about 14% to about 38% basic isoforms as detected by iCIEF.

[0404] In some aspects, the rat hybridoma cells in the MCB composition or WCB composition are YB2 / 0 cells. In some aspects, the recombinant protein to be produced from the MVB or WCB composition is a monoclonal antibody. In some aspects, the monoclonal antibody is an anti-CD20 antibody.

[0405] In some aspects, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0406] Also provided are methods of making a recombinant protein by using an MCB or WCB composition as disclosed herein. In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody is an anti-CD20 antibody. In some aspects, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 7; and a light chain having the amino acid sequence shown in SEQ ID NO: 9.

[0407] Recombinant proteins and compositions

[0408] A recombinant protein manufactured according to any method disclosed herein is provided. In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody is an antibody that binds to an epitope of CD20. In some aspects, the monoclonal antibody is TG-1101 (TG Therapeutics, Inc.) or an antibody that binds to the same epitope as TG-1101. In some aspects, the monoclonal antibody is TG-1101.

[0409] In one aspect, the disclosure provides compositions comprising recombinant proteins produced in rat hybridoma cells (e.g., YB2 / 0) by any method disclosed herein. In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody targets the CD20 antigen (i.e., an anti-CD20 antibody). In some aspects, the monoclonal antibody is TG-1101 or an antibody that binds to the same epitope as TG-1101. In some aspects, the monoclonal antibody is TG-1101.

[0410] In some aspects, the compositions (e.g., comprising anti-CD20 antibodies) produced by the methods disclosed herein have a unique glycosylation / N-glycan profile characterized by, e.g., between about 20% and about 40% fucosylated glycans and / or between about 10% and about 20% galactosylated glycans.

[0411] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising one or both of: i. about 10 to 20% galactosylated glycans; and / or ii. about 23 to 36% fucosylated glycans.

[0412] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising about 10 to 20% galactosylated glycans and about 23 to 36% fucosylated glycans.

[0413] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising about 36% fucosylated glycans.

[0414] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising about 16 to 18% galactosylated glycans.

[0415] In some aspects, the N-glycan profile includes about 17% galactosylated glycans.

[0416] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising at least about 10% bisecting N-glycans. In some aspects, the N-glycan profile comprises about 12% to 30% bisecting N-glycans. In some aspects, the N-glycan profile comprises about 18% bisecting N-glycans.

[0417] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising less than 5% sialylated glycans. In some aspects, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some aspects, the N-glycan profile comprises an undetectable amount of sialylated glycans.

[0418] In some aspects, the monoclonal antibody comprises an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans. In some aspects, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some aspects, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some aspects, Man5 N-glycans are the only high mannose species in the N-glycan profile.

[0419] In some aspects, the cell culture composition comprising a recombinant protein, a monoclonal antibody, an anti-CD20 antibody, or TG-1101 produced in rat hybridoma cells by any of the methods disclosed herein is a pharmaceutical composition, wherein the recombinant protein, the monoclonal antibody, the anti-CD20 antibody, or TG-1101 is formulated with a pharmaceutically acceptable carrier.

[0420] As used herein, the phrase "pharmaceutical composition" refers to a composition suitable for pharmaceutical administration, such as administration to humans. Such compositions may include substances that are impurities at levels not exceeding acceptable levels for pharmaceutical administration (such levels include the absence of such impurities), and may include, for example, pharmaceutically acceptable excipients, vehicles, carriers, and other inactive ingredients, in addition to any active agent(s) to formulate such compositions for ease of administration.

[0421] In some aspects, the pharmaceutical composition comprises one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid.

[0422] Also provided is a method for treating a hematological malignancy in a subject in need thereof by administering a recombinant protein or monoclonal antibody (e.g., anti-CD20 antibody) manufactured according to any method disclosed herein. In some aspects, hematological cancer is lymphoma, leukemia, or myeloma. In some aspects, hematological cancer is selected from B cell lymphoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom macroglobulinemia (WM), diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), Burkitt lymphoma (BL), Richter transformation, or primary central nervous system lymphoma (PCNSL).

[0423] Also provided are methods of treating an autoimmune disorder in a subject in need thereof by administering a recombinant protein or monoclonal antibody (eg, an anti-CD20 antibody) produced according to any of the methods disclosed herein. In some aspects, the autoimmune disease is multiple sclerosis, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic or xenogeneic transplantation (organs, bone marrow, stem cells and other cells and tissues), transplant rejection, graft-versus-host disease, lupus erythematosus, inflammatory diseases, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's thyroiditis and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases, or uveitis.

[0424] In some aspects, the autoimmune disease is multiple sclerosis. In some aspects, multiple sclerosis is a relapsing form of multiple sclerosis. In certain embodiments, the relapsing form of multiple sclerosis is clinically isolated syndrome (CIS); relapsing-remitting MS (RRMS); active secondary progressive MS (SPMS); or primary progressive MS (PPMS). In preferred aspects, the subject is a human.

[0425] The following examples are offered by way of illustration and not by way of limitation.

[0426] Example

[0427] In the following examples, the cell culture process used to control the growth and productivity of the anti-CD20 IgG1 monoclonal antibody (TG-1101) expressed by YB2 / 0 rat hybridoma cells is exemplified by, for example, the selection of cell culture medium (Example 1), culture pH, temperature and pCO2 control methods (Example 2). Example 3 illustrates the 15,000L commercial scale production of anti-CD20 antibodies in rat hybridoma cells. Example 4 illustrates the detection and removal of viruses or other foreign substances in a 15,000L commercial scale production process. Example 5 illustrates the calculated pH process parameter "Integrated pH2 Difference" and its effect on harvest titer, fucosylation percentage and integrated viable cell density (IVCD). Finally, Example 6 illustrates the unique glycosylation profile of the anti-CD20 antibodies produced by the methods disclosed herein.

[0428] Resulting in increased process productivity, while also reducing protein fucosylation levels and increasing the Fc effector activity of the antibody. The process shown for controlling both productivity and product quality was shown using this unique, uncommon mammalian expression system. The observed results were unexpected and surprising - although YB2 / 0 cells have been shown to support low levels of fucosylation (Kanda, Y. et al., Biotechnol Bioeng 94: 680-688 (2006)), these cells have not demonstrated that fucosylation levels can be lowered and productivity can be higher by cell culture process means alone. In addition, the production of anti-CD20 antibodies in rat hybridoma cells on a commercial scale has never been shown, let alone anti-CD20 antibodies with unique glycosylation characteristics.

[0429] Example 1: Increasing the productivity of anti-CD20 antibodies expressed by the YB2 / 0 rat hybridoma cell line by selection of cell culture medium

[0430] Materials and methods

[0431] Cell culture

[0432] The YB2 / 0 rat hybridoma cell line (ATCC CRL 1662) was adapted to 10 commercially available cell culture media (Table 2). After scale-up from thawing in static T-flasks in EM-SF2-P5-H4 medium, cells were passaged into 125 mL shake flasks (at 2-8°C) containing each of the 10 new culture media and placed in a humidified incubator at 37°C with agitation at 90 RPM and % CO2 corresponding to the manufacturer's recommended values ​​for each culture medium. Cultures were passaged daily with a target seeding density of 0.5 x 10 6, until cell viability drops below 60% (in which case culture is terminated), or until growth rate activity stabilizes for 5 days, after which cells are considered adapted. To determine the best passaging strategy, growth curves were drawn based on the best performing culture medium. All culture media were supplemented with 1 mL / L of 1000X cholesterol and 6 mM L-glutamine.

[0433] Table 2: Basal medium for evaluating the growth of YB2 / 0 cell line

[0434] Basic culture medium supplier Hycell Medium GE IS CHO-SD G10.6Medium Irvine Scientific CD Hybridoma Thermo Fisher PFHM-IIProtein Free Hybridoma Thermo Fisher Ex-Cell CD Hybridoma SAFC UltraDOMA-PF Hybridoma Lonza ProDOMA 1 Lonza IS MAB-CD Irvine Scientific LM|CDM4Mab Cytiva / GE ActiCHO-P GE

[0435] After a media adaptation period in 125 mL shake flasks, it was determined that cells had successfully adapted to four new media: CDM4Mab, CD Hybridoma, Hycell, and ProDoma 1. To obtain seven unique basal media conditions for further screening against six commercially available feeds (Table 3), the media that performed best in terms of growth and viability were selected. Mixed with 3 other successful media in a 50:50 ratio. A definitive screening experimental design (DOE) was used to identify the best performing combination of growth and feed media when run in fed-batch mode using the Ambr15 cell culture platform (Sartorius). In addition, 1 vessel was run with historical basal medium and fed as a control and 5 vessels were run with the best performing new media supplemented with different concentrations of both fatty acid supplements and Long R3 IGF-1 supplement Run. The process parameters for all vessels are shown in Table 4. The measured output for each parameter was viable cell density, cell viability, antibody titer, and glycosylation profile of the expressed mAb for the first 12 conditions (including controls), on the day of harvest.

[0436] Table 3: Feed media for evaluating growth and productivity of YB2 / 0 cell line

[0437]

[0438] Table 4: Cell culture process conditions used to screen different cell culture media

[0439]

[0440]

[0441] Protein analysis features

[0442] N-Glycans

[0443] N-linked glycans are cut from the product by enzymatic deglycosylation using PNGase F and labeled with 2-aminobenzamide (2-AB), a fluorescent compound. Labeled glycans are resolved using a hydrophilic interaction partitioning mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360nm and emission at 428nm). Blanks, glycans from reference standards, and glycan standards (human IgG N-linked glycan library) are also injected to assess system suitability. Peak identification from the resulting test sample chromatogram is based on retention time and is identified relative to the peak in the glycan standards confirmed by mass spectrometry.

[0444] ADCC potency assay

[0445] The ADCC assay is a cell-based assay using CD20-expressing Jeko-1 cells, a human mantle cell lymphoma cell line, and Eurofins-DiscoverX "KILR CD16a effector cells" (which are human CD8+ T lymphocytes derived from a single donor engineered to express CD16 (FcγRIII) on their plasma membrane surface). Target cell lysis mediated by the antibody samples was measured. Dose-response curves were modeled using 4-parameter logistic regression (4PL), and results were reported as % efficacy relative to a reference standard.

[0446] CDC potency assay

[0447] The CDC assay is a cell-based assay using Jeko-1 cells expressing CD20 and rabbit serum as a source of complement. CDC-mediated cell lysis is measured. The dose-response curve is modeled using the 4PL equation and the results are reported as % potency relative to a reference standard.

[0448] CD20 binding activity

[0449] In the CD20 binding activity assay, the binding of anti-CD20 antibodies to the CD20-expressing human mantle cell lymphoma cell line Jeko-1 was evaluated using electrochemiluminescence (Meso Scale Discovery). The dose response curve was modeled using the 4PL equation and the results were reported as % potency relative to the reference standard.

[0450] FcγRIIIa binding

[0451] Binding of anti-CD20 antibody samples to FcγRIIIa was evaluated by surface plasmon resonance (SPR). The method followed a direct binding assay methodology, where the Fc receptor (ligand) was immobilized directly on an appropriate flow cell on the surface of a sensor chip, and the analyte was injected onto the chip to assess binding. The high affinity allele of FcγRIIIa (158V) was determined.

[0452] The equilibrium dissociation constant (KD) and relative affinity of each sample relative to the reference standard were determined for each receptor. The rate of change of the SPR signal was analyzed using a 1:1 Langmuir model to generate apparent rate constants for the association and dissociation phases of the reaction as well as the equilibrium dissociation constant. The results are reported as the potency % for the FcγRIIIa-158V variant relative to the reference standard.

[0453] C1q binding

[0454] Binding of anti-CD20 antibodies to C1q was evaluated using a C1q ELISA-based approach. Dose-response binding was modeled by weighted nonlinear regression using a 4-parameter logistic fit and results are reported as % potency relative to a reference standard.

[0455] result

[0456] After successfully adapting the cells to the new medium, the adapted cells were used to inoculate an Ambr 15 bioreactor (Sartorius). The purpose of this study was to identify a growth medium and feed medium combination that produced approximately twice the titer and similar product quality of a fed-batch process (~320 mg / L, 40-60% fucosylation) using PROEMS-02 growth medium and Cell Boost 3 feed medium. This study was performed using the process conditions shown in the above method.

[0457] A wide range of cell growth performance was observed in different growth medium and feed medium combinations (Table 5), with the integral of the viable cell density (IVCD) results on day 10 ranging from 10-87 x 10 6 viable cells / mL / day ( Figure 2 ), and the cell viability ranged from 7.5-93.5% on day 10 ( Figure 3 ). Figure 4 Describe the top 11 media conditions in terms of harvest titers (plus control condition: PRO-EMS-02 / Cell Boost 3), and Figure 5The percent (%) fucosylation results for the top 11 media conditions plus the control are depicted. All 11 conditions outperformed the control media in terms of titer, and CDM4Mab was the best represented of the top 11 new growth media and feed media combinations. The combination of CDM4Mab media with BalanCD CHO Feed 4 produced the highest titer of 0.563 g / L and represented the only media / feed combination that came close to achieving a 2-fold increase in titer.

[0458] In terms of product quality, CDM4Mab / BalanCD CHO Feed 4 showed significantly lower % fucosylation (~24%) compared to the range supported by the control conditions (i.e., 40-60%). In addition, when CDM4Mab growth medium was mixed with CDHybridoma medium in a 50:50 ratio and combined with the feed medium BalanCD CHO Feed 4, the results were still the best performing condition in terms of titer (~0.38g / L), while the overall % fucosylation increased to ~72%. This shows that while keeping the feed medium consistent (BalanCD CHO Feed 4), the % fucosylation results on the mAb can be changed by changing the ratio of the different growth media.

[0459] Table 5: Evaluated cell culture medium and feed medium combinations

[0460]

[0461]

[0462] The cell culture growth and feed media identified in the above studies were subsequently used for clinical GMP manufacturing of TG-1101 (anti-CD20 IgG1 monoclonal antibody). The API materials derived therefrom were tested for % fucosylation and Fc effector function / activity. The control media (PROEMS-02 growth medium and Cell Boost 3 feed medium) were used relative to the new media ( Growth Media and BalanCD CHO Feed A comparison of mAb characterization results produced in feed medium (with or without feed medium) is shown below: Figure 6 Fucosylation%, Figure 7 For FcRIIIa-158V binding, Figure 8 For CD20 binding, Fig. 9 For ADCC activity, Fig.10 For C1q binding and Fig.11for CDC activity. The results showed a significant decrease in % fucosylation, associated with a significant increase in FcγRIIIa-158V binding, and a modest increase in ADCC activity. There were no changes in CD20 antigen binding due to switching cell culture media. In addition, there were no changes in C1q binding, and little to no change in CDC activity. Collectively, these results suggest that by changing the cell culture medium used for this rarely used YB2 / 0 rat hybridoma cell line, the function and activity of the expressed antibody can be adjusted by means of the cell culture medium alone. There are no literature reports of this relationship for YB2 / 0 rat hybridoma cells in the public domain.

[0463] in conclusion

[0464] In this example, a method for increasing process performance (cell growth, recombinant protein productivity) and adjusting protein glycosylation by a cell culture process using YB2 / 0 cells is shown. In this example, it was found that cell growth and feed medium have important effects on protein glycosylation profiles. The combination of growth and feed medium ( BalanCD CHO Feed ) supported a significant reduction in percent fucosylation levels, which upon further testing was found to support an increase in potency of binding via FcγRIIIa.

[0465] Example 2: Increasing the productivity of anti-CD20 antibodies expressed by YB2 / 0 rat hybridoma cell line by cell culture pH, temperature and pCO2 control methodology

[0466] Materials and methods

[0467] Cell culture

[0468] The YB2 / 0 rat hybridoma cell line (ATCC CRL 1662) genetically engineered to stably express the anti-CD20 IgG1 monoclonal antibody TG-1101 was thawed from cryopreservation and cultured in CDM4Mab (GE Healthcare) chemically defined growth medium at 37°C. After a series of passages to culture vessels of increasing volume, and after achieving sufficient cell biomass, the cells were inoculated into a 10 L laboratory scale bioreactor. The process conditions utilized during these cultures are shown in Table 6.

[0469] The 10 L bioreactor was equilibrated after cell culture medium addition. Culture medium was added to the initial volume target. Dissolved oxygen (DO) and pH probes were calibrated prior to use. Initial bioreactor set points included temperature, pH, dissolved oxygen, and agitation rate. The bioreactor was inoculated with cell culture from a seed train expansion vessel at 0.5 x 10 6 The target inoculation of viable cell density was 100 viable cells / mL. During the production bioreactor process, pH was controlled by alkali addition and CO2 sparging as needed. Dissolved oxygen was controlled by oxygen and air sparging as needed. Defoamers were added as needed to alleviate foaming problems. Offline pH, pCO2 / pO2, osmotic pressure and metabolites were monitored daily using a blood gas analyzer (Siemens), and viable cell density (VCD) and vitality were monitored using a ViCell automatic cell counter (Beckman Coulter). The process metabolites of daily samples including glucose, lactate, glutamine, ammonia and osmotic pressure were also measured using a NovaBioprofile FLEX2 analyzer (Nova Biomedical).

[0470] Throughout the bioreactor culture, various feed media and solutions are added to support the nutritional needs of the cells. BalanCD CHO Culture medium (Irvine Scientific) was added at a specified volume (4.0% of the initial bioreactor working volume) on days 3, 5, 7, and 9. The calculated volume of concentrated glucose solution (to 4.0 g / L) was fed when the daily bioreactor sample measurement was <3.0 g / L on a specified process day. A fixed volume of glutamine solution was fed as a bolus on day 3, and the calculated volume (to 4.0 mM) was fed when the daily bioreactor sample measurement was <3.00 mM on a specified process day. 1000X concentrated cholesterol lipid solution was added to the culture as a fixed bolus addition on days 0 and 4 (0.256% v / v).

[0471] The process set points were shifted at fixed times throughout the bioreactor culture. The culture pH set point was shifted from 7.10 + / - 0.30 to a different pH set point on process day 3 (Table 6). The temperature set point was shifted from 37°C to 35°C on process day 1 and again from 35°C to 32.5°C on process day 3.

[0472] Production bioreactors are harvested based on either culture duration or cell viability criteria, whichever occurs first. Cell culture harvests are clarified to remove cells and then protein A purified for measurements of product quality (SEC, iCIEF, N-linked glycans) and functional testing, including both effector function and binding assays.

[0473] Table 6: Cell culture process conditions for laboratory-scale bioreactors

[0474]

[0475]

[0476] a Values ​​reflect both the process set point + / - the deadband value (i.e. the range in which the pH is free to move centered on the process set point)

[0477] Protein analysis features

[0478] N-Glycans

[0479] N-linked glycans are cut from the product by enzymatic deglycosylation using PNGase F and labeled with 2-aminobenzamide (2-AB), a fluorescent compound. Labeled glycans are resolved using a hydrophilic interaction partitioning mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360nm and emission at 428nm). Blanks, glycans from reference standards, and glycan standards (human IgG N-linked glycan library) are also injected to assess system suitability. Peak identification from the resulting test sample chromatogram is based on retention time and is identified relative to the peak in the glycan standards confirmed by mass spectrometry.

[0480] SEC

[0481] The SEC-HPLC method is used to determine the molecular size distribution and purity of IgG1 antibody. SE-HPLC separates proteins based on hydrodynamic radius, and adopts the TSKgelG3000SWXL post (Tosoh) designed to be separated in the protein of the scope of 10,000 to 500,000Da. Gained chromatogram is monitored at 214nm, peak is integrated, and the amount of each peak (monomer, dimer, aggregation and fragment) is described with the percentage of its area. Sample, reference standard, molecular weight standard and blank injection are carried out to evaluate the suitability of system.

[0482] iCIEF

[0483] Imaging capillary electrophoresis (iCIEF) is used to evaluate and quantify the distribution of charge-based isotypes of TG-1101 using Protein Simple iCE3 analyzer. The test sample solution includes 0.15% methylcellulose, 1.2M urea, 8% ampholytes pH 3-10.5 and pH 10.10 and 8.40pH markers. Focus on 1500V for 1 minute and then 3000V for 8 minutes. The results reported include the combined values ​​of the pI of the main peak, the % peak area of ​​the main peak, and the % peak area of ​​the alkaline peak and the acidic peak. Blank samples and hemoglobin controls are also run to evaluate system suitability.

[0484] ADCC potency assay

[0485] The ADCC assay is a cell-based assay using CD20-expressing Jeko-1 cells, a human mantle cell lymphoma cell line, and Eurofins-DiscoverX "KILR CD16a effector cells" (which are human CD8+ T lymphocytes derived from a single donor engineered to express CD16 (FcγRIII) on their plasma membrane surface). Target cell lysis mediated by the antibody samples was measured. The dose-response curve was modeled using the 4PL equation, and the results were reported as % potency relative to the reference standard.

[0486] CDC potency assay

[0487] The CDC assay is a cell-based assay using Jeko-1 cells expressing CD20 and rabbit serum as a source of complement. CDC-mediated cell lysis is measured. The dose-response curve is modeled using the 4PL equation and the results are reported as % potency relative to a reference standard.

[0488] CD20 binding activity

[0489] In the CD20 binding activity assay, the binding of anti-CD20 antibodies to the CD20-expressing human mantle cell lymphoma cell line Jeko-1 was evaluated using the Meso Scale Discovery (MSD) method. The dose response curve was modeled using the 4PL equation and the results were reported as % potency relative to the reference standard.

[0490] FcγRIIIa-158V binding:

[0491] Binding of anti-CD20 antibody samples to FcγRIIIa was evaluated by surface plasmon resonance (SPR). The method followed a direct binding assay methodology, where the Fc receptor (ligand) was immobilized directly on an appropriate flow cell on the surface of a sensor chip, and the analyte was injected onto the chip to assess binding. The high affinity allele of FcγRIIIa (158V) was determined.

[0492] The equilibrium dissociation constant (KD) and relative affinity of each sample relative to the reference standard were determined for each receptor. The rate of change of the SPR signal was analyzed using a 1:1 Langmuir model to generate apparent rate constants for the association and dissociation phases of the reaction as well as the equilibrium dissociation constant. The results are reported as the potency % for the FcγRIIIa-158V variant relative to the reference standard.

[0493] C1q binding:

[0494] Binding of anti-CD20 antibodies to C1q was evaluated using a C1q ELISA-based method. Dose-response binding was modeled by weighted nonlinear regression using a 4-parameter logistic fit and results are reported as % potency relative to a reference standard.

[0495] result

[0496] A retrospective analysis of manufacturing and bench-scale cell culture of the YB2 / 0 rat hybridoma cell line was performed to analyze trends in process performance data. One of the most significant trends in the data was the positive relationship between cell growth and cumulative culture exposure to pCO2 ( Fig.12 In general, cultures with high cumulative cell growth (IVCD) also had lower cumulative pCO2 exposure (integrated pCO2). Another important trend in the data is the positive relationship between harvest titer and cumulative culture exposure to pCO2 ( Fig.13 ). In general, cultures with high harvest titers of monoclonal antibodies also had lower integrated pCO2. Further investigation of these relationships was performed. Evaluation was performed on 10 L bench-scale cultures, specifically testing the use of different pH control strategies to promote changes in pCO2.

[0497] Production bioreactor cultures were performed at bench scale to evaluate different post-shift pH set points and control ranges. Cell growth results are shown in Fig.14 , and shows the pH dependence effect on the observed results. At the lowest post-conversion pH control range studied (6.70 + / - 0.10), the peak viable cell density (VCD) was the lowest among the conditions tested (18.9 x 10 6Cells / mL). Conversely, at the highest pH control range studied (6.95+ / -0.05), the peak VCD was the highest among the tested conditions. The pH control range between these two ranges showing cell growth and peak VCD results was moderate in nature. The results emphasize that as the process pH control range increases, there is a significant increase in observed cell growth. Lower cell growth with lower culture pH has been seen previously in CHO cells (Trummer, E. et al., Biotechnology and Bioengineering 94: 1033-1044 (2006)). However, this behavior has never been reported for the YB2 / 0 cell line.

[0498] The cell viability results also showed trends under different pH control ranges and showed Fig.15 At the lowest pH control range studied, cell viability declined earlier, triggering a bioreactor harvest on process day 13. At the higher pH control range studied, cell viability declined much more slowly, resulting in a bioreactor harvest on process day 14. These results are significant because it shows that for this unique cell line, controlling pH alone is a process means by which culture life can be increased. In addition to this extended culture life, the resulting impact on harvest titer is also significant.

[0499] Glucose is consumed by mammalian cells as the primary source of energy and carbon necessary for oxidative metabolism. The major waste byproduct from glucose metabolism in mammalian cells is lactic acid. High levels of lactic acid have previously been shown to be inhibitory to process performance (Hassell, T. et al., Appl Biochem Biotechnol. 30:29-41 (1991)). Glucose and lactic acid levels in the cell culture medium were monitored in each culture ( Fig.16 , Fig.17 ). Glucose level is similar in each cultivation, and owing to concentrated glucose feed solution being optionally added into each cultivation and not exhausted.Yet, from the 9th day to the observed lactic acid level during results, there is major difference.Relatively low pH process condition supports higher lactic acid level, pH 6.70+ / -0.10 condition supports the highest final lactic acid level of 6.8g / L, and pH 6.95+ / -0.05 condition supports the minimum final lactic acid level of 2.2g / L.These results show, for YB2 / 0 cell, there is major difference in the metabolism as the function of process pH control, and for this unique cell line this is the first time to report this relationship.

[0500] Dissolved carbon dioxide levels were also monitored in each individual cell culture condition ( Fig.18). It has been previously shown with other mammalian cell lines that high levels of pCO2 adversely affect overall process performance (deZengotita, V., et al., Cytotechnology 28: 213-227 (1998)). The pCO2 level observed in the process during bioreactor conditions is a function of the pH set point, the level of lactic acid in the culture (higher lactic acid levels support a more acidic environment) and the overall air and oxygen injection rate in the bioreactor. In each 10L bioreactor culture evaluated, the same maximum air injection rate was used. The oxygen injection rate was controlled by the bioreactor controller, and the culture with higher VCD levels usually requires higher flow rates. The YB2 / 0 cell culture results show that the lowest pH control conditions support the highest pCO2 levels observed. That is, pH 6.70+ / -0.10 culture leads to a peak pCO2 level of 146mmHg in the mid-term of the culture, while higher pH control conditions all support pCO2 levels <100mmHg. Collectively, these results highlight how higher pH controlled conditions support the lowest pCO2 levels, as well as the highest cell growth profiles and antibody titers as discussed below.

[0501] Antibody titers were monitored in each of the above laboratory-scale cell culture conditions ( Fig.19 ). In all pH control ranges evaluated, antibody titers began to increase between process days 4-5. Antibody titers similarly increased to approximately the same level until day 10, after which they began to diverge. The higher pH culture conditions continued to increase until the harvest point, with the following harvest day titers measured: 1.26 g / L for pH condition 6.85+ / -0.05, and 1.19 g / L for pH condition 6.95+ / -0.05 g / L. The lower pH culture conditions did not show a large titer increase after day 10, resulting in a harvest titer of 1.0 g / L for pH condition 6.80+ / -0.03 and a harvest titer of 0.79 g / L for pH condition 6.70+ / -0.10. The collective results show that as the pH control range increases to higher values ​​after the transition, pCO2 decreases and with Fig.13 The behavior highlighted in was similar, with an increase in antibody productivity.

[0502] These results are surprising because it has been previously found in other cell lines that an increase in culture pH supports a decrease in productivity (Jiang, R. et al., Bioprocess Biosyst Eng 41: 1731-1741 (2018)), and a decrease in culture pH supports an increase in productivity (Seo, JS et al., Appl Microbiol Biotechnol. 97: 5283-5291 (2013)). In other reports on CHO cell lines, the results are mixed. In two reports, an increase in recombinant productivity was seen at higher pH values ​​but in the pH range of 7.00-7.20 (Yoon, SK et al., Biotechnol. Bioeng. 89: 345-356 (2005); Kim, HS et al., J Microbiol and Biotech. 17: 712-720 (2007)). In another report, it was found that the culture pH had no effect on productivity (Hennicke, J. et al., New Biotechnology 50:20-26 (2019)). In a different report on a CHO cell line expressing an antibody, it was found that pH 6.8 supported 2X higher titers compared to pH 7. (Oguchi, S. et al., Animal Cell Technology; Basic & Applied Aspects 13:169-172 (2003)). It is believed that there are no available reports describing the relationship between culture pH and YB2 / 0 cells, and this work is believed to be the first documented example.

[0503] The expressed antibody (TG-1101) was protein A purified from each 10L bioreactor culture and analyzed for product quality, including N-linked glycans (protein glycosylation), charge heterogeneity (iCIEF), and size (SEC). Table 7 summarizes the N-linked glycans and highlights how the highest pH control range (6.95+ / -0.05) supports the lowest level of fucosylation % (18.4%). In addition, as the pH control range increases (i.e., 6.80+ / -0.03 and higher), the total amount of sialylated N-glycans increases from 0% to 1%, effectively introducing new N-glycan species into the spectrum. Therefore, for this unique YB2 / 0 cell line, protein fucosylation levels can be nominally controlled by pH-mediated pCO2 control. Table 8 summarizes the charge heterogeneity results, which were not significantly affected by different pH and pCO2 conditionality. Table 9 summarizes the size characteristics results of the antibodies and emphasizes that individual species do not change in different pH and pCO2 conditions.

[0504] Table 7: Protein glycosylation results of cultures evaluated at different pH control ranges (after conversion)

[0505] pH control range Fucosylation (%) Sialylation (%) G0(％) G0B(%) G1(％) 6.70+ / -0.10 24.6 0 50.7 8.9 9.7 6.80+ / -0.03 27.5 0.8 47.1 7.8 8.3 6.85+ / -0.05 24.9 0.8 45.9 6.5 11.5 6.95+ / -0.05 18.4 0.3 46.7 7.0 11.8

[0506] Table 8: iCIEF results evaluating cultures with different pH control ranges (after shift)

[0507] pH control range Acidic substances (%) main(%) Alkaline substances (%) 6.70+ / -0.10 22.8 56.9 20.4 6.80+ / -0.03 22.9 59.3 18.0 6.85+ / -0.05 25.2 58.5 16.3 6.95+ / -0.05 28.2 52.8 19.1

[0508] Table 9: SEC results evaluating cultures with different pH control ranges (after shift)

[0509] pH control range monomer(%) Dimer (%) Aggregate (%) Fragment (%) 6.70+ / -0.10 98.9 1.1 0 0 6.80+ / -0.03 98.9 1.1 0 0 6.85+ / -0.05 98.3 1.6 0 0.2 6.95+ / -0.05 98.4 1.3 0 0.3

[0510] Purified antibodies (TG-1101) from each 10L bioreactor culture were also tested in various binding assays (CD20 antigen, Fcγ receptor, complement component 1q) and Fc effector activity assays (ADCC, CDC). Results from each of these tests are shown in Table 10. Fc RIIIa-158V, CD20 and C1q binding results were comparable in each culture condition evaluated. ADCC and CDC bioassay results were also shown to be comparable in each culture condition. Collectively, these results emphasize that higher cell growth and antibody titer caused by higher process pH control and associated lower pCO2 control will not adversely affect the functional activity of the antibody.

[0511] Table 10: Binding / Effect Function Test Results Evaluating Cultures in Different pH Control Ranges (After Shift)

[0512]

[0513]

[0514] a Results are expressed relative to the reference standard used for each assay.

[0515] in conclusion

[0516] In this example, the inventors show another way to increase process performance (cell growth, recombinant protein productivity) and how to adjust protein glycosylation by a cell culture process using the YB2 / 0 cell line. In this example, the process parameter of cumulative pCO2 exposure during cell culture was found to have an important effect on both cell growth and antibody productivity. Culture pH was confirmed to be the main driver for culture pCO2 levels. It was found that for this unique and uncommon mammalian expression system, both the higher pH control range (>6.80) and the control of low pCO2 levels (<100 mmHg) in culture supported high process titers (≥1.0 g / L). Successful demonstration of control of fucosylation % levels and the resulting binding and effector function / activity of the expressed anti-CD20 antibody were shown under high bioreactor productivity conditions.

[0517] Example 3: Commercial-scale manufacturing process for producing TG-1101 in YB2 / 0 rat hybridoma cells

[0518] In this example, the manufacturing process of TG-1101 (TG Therapeutics, Inc.) expressed in YB2 / 0 rat hybridoma cells at 15,000 L is described. An overview of the manufacturing process of TG-1101 is illustrated in the flow chart of Table 12.

[0519] Table 12: TG-1101 manufacturing process flow chart

[0520]

[0521]

[0522] In summary, the production of each batch of TG-1101 begins with the thawing of working cell bank (WCB) vials, which is further described below. Culture is amplified by a series of shake flasks and seed bioreactors to meet the inoculum requirements of a 15,000L production bioreactor operated in fed-batch mode. The bioreactor is harvested and clarified by centrifugation and then deep filtration. The clarified harvest is purified by three chromatographic steps including protein A, cation exchange and anion exchange, aiming at purifying TG-1101 and reducing process impurities such as host cell proteins and residual DNA. Purification process (as described in Example 4) includes the step of ensuring virus safety, including virus inactivation (solvent / detergent) and virus filtration step. Final UFDF and formulation steps are used to concentrate TG-1101 and buffer exchange into formulation buffer and reach the desired product concentration. The ready to fill drug substance was formulated to obtain a concentration of 25.0 mg / mL of TG-1101 in 25 mM sodium citrate, 154 mM sodium chloride, 0.07% polysorbate 80, pH 6.5. After filling, the TG-1101 drug substance (DS) was frozen at ≤-60°C and then stored frozen at ≤-35°C.

[0523] Additional description of upstream and downstream process operations is provided below.

[0524] Expression vectors, production cell lines and cell banks

[0525] The host cell line used to generate the cell line producing TG-1101 was the rat hybridoma cell line YB2 / 0. After transfection of the expression vector HK463-25 (containing the immunoglobulin heavy and light chain cDNA sequences of TG-1101) into the YB2 / 0 host cell line, the production cell line R603-12D11 was developed. FIG. 20 depicts a map of the expression vector of HK463-25 producing TG-1101 in a 15,000L bioreactor.

[0526] Expression vector

[0527] The expression vector HK463-25 includes various elements optimized for stable expression in YB2 / 0 host cell lines. The Rous sarcoma virus long terminal repeat (RSV LTR) promoter is used for constitutive expression of both heavy chain and light chain cDNA. The promoter corresponds to the long terminal repeat of RSV genome, contains enhancer elements in its 5' region and has strong transcriptional activity in YB2 / 0 cell lines. Transcription termination and polyadenylation of both heavy chain and light chain cDNA are provided by human growth hormone polyadenylation sequence (hGH polyA). Chimeric introns are introduced into the 5' of the cDNA sequence of each antibody chain to improve expression. The intron is optimized for splicing and is composed of 5' donor sequences from human beta-globin and 3' receptor sequences from Ig heavy chain variable genes. Beta-lactamase gene confers ampicillin resistance (AmpR), and is provided so that it is possible to realize the production of plasmid in Escherichia coli. The enzyme neomycin-phosphotransferase II (NeoR) is under the control of the SV40 promoter and confers resistance to the antibiotic G418 to the transfected cell lines, thus serving as a selectable marker. Dihydrofolate reductase (Dhfr) is under the control of the SV40 promoter and confers resistance to methotrexate (MTX), and can also serve as a selectable and amplifiable marker in transfected cell lines.

[0528] The HK463-25 expression vector (Figure 20) is 11.1 kb in size and contains 5 open reading frames of the antibody heavy chain, light chain, Dhfr, NeoR and AmpR genes in the same orientation. The restriction sites shown in the figure are used for Southern blot analysis of the integration construct. The unique NotI restriction site located 3' of the NeoR gene is used for linearization of the vector before transfection.

[0529] Production cell line R603-12D11

[0530] The production cell line R603-12D11 was generated after transfection of the host cell line, selection and screening of the transfectants, and then limited dilution cloning. The clones were screened, the production cell line R603-12D11 was selected, and adapted to serum-free medium. A pre-seed stock (PSS) cell bank was prepared. An overview of the steps involved in the generation of the production cell line R603-12D11 is shown in Table 13.

[0531] Table 13: Production cell line R603-12D11 generation

[0532]

[0533] A cryovial of YB2 / 0 cell bank (YB2 / 0-301 04 / 147) was thawed and diluted to 1 × 10 every 3 to 4 days with fresh medium (EMS medium containing 5% FCS). 5 Grow cells at a cell density of 10 cells / mL. On the day before transfection, grow cells at a density of 2×10 5 The cells were seeded at a density of 10 cells / mL to allow exponential phase to be reached before transfection. 44.5 μg of NotI-linearized expression vector HK463-25 ( FIG. 20 ) was transfected into 5×10 cells / mL by electroporation using animal component-free Optimix reagent (Equibio). 6 Cells were diluted in culture medium and seeded in 96-well plates at 100 cells / well. Three days after electroporation, selection was started with 1 g / L G418 in culture medium.

[0534] After selection in G418 medium, transformants were screened for titer by ELISA. More than 3000 transformants were screened, and more than 200 best production wells were selected for further testing and for continuous passage. A second titer screening was performed to further reduce the number of clones. The screening was then performed by ELISA for additional screening of antibody fucose levels. In order to provide the expected level of CD16 activation of the antibody, a fucose level lower than 40% was considered ideal; CD16 activation is inversely proportional to the fucose level. Further screening was performed using a cell-based CD16 activation assay, in addition to evaluating the free κ / IgG ratio (<0.2, for purification), IL-2 secretion was also evaluated, resulting in a reduction to a total of 5 cell lines for further development.

[0535] 5 candidate cell lines were cloned by limiting dilution at 0.4 cells / well in EMS culture medium containing 5% FCS. Screening was performed for cloned IgG productivity, fucose level, CD16 activation and free κ / IgG ratio to identify production cell line R603-12D11. As the selected cell line was expanded from the cloning step, it was also converted to serum-free culture medium and rescreened before preparing the small cell bank to ensure the desired phenotype. The small cell bank was then thawed and expanded to generate R603-12D11 seed pre-stock cell bank (PSS). Before generating MCB, R603-12D11PSS cell bank was shown to be free of mycoplasma, foreign viruses and microbial contamination.

[0536] Characterization of the master and working cell banks derived from the R603-12D11 producer cell line included testing for identity, genotypic and phenotypic characteristics, and for the presence of adventitious materials.

[0537] Master Cell Bank (MCP) Preparation and Testing

[0538] MCB is manufactured at Henogen (later acquired by NovaSep). MCB lot number G071 / MCB / 070208 is prepared by thawing and expanding a vial of the production cell line R603-12D11 seed stock in serum-free medium EM-SF2 P500 H4 (EMS basal medium supplemented with 2-mercaptoethanol, ethanolamine, NaHCO3, ferric citrate, pluronic acid, HEPES and recombinant human insulin). The cells were expanded in T-flasks and roller bottles for eleven days. The cell suspension was concentrated by centrifugation and aliquoted into 13 individual fractions. Each fraction was centrifuged and resuspended to 10×10 6 The suspension was then aliquoted into 18 cryovials per fraction, yielding a total of 234 MCB cryovials. The cryovials were placed on dry ice and then placed in a cryobox, which was placed in a -80°C freezer for 21 hours. The cryovials were transferred to liquid nitrogen tanks for long-term storage on February 19, 2007 and are currently stored at multiple locations. The cell generation number from PSS to MCB for the production cell line is 10.4.

[0539] After generating MCB batch number G071 / MCB / 070208, MCB was directly tested, as well as further characterized by testing the WCB derived therefrom. The identity test of MCB was performed; the test results confirmed that the MCB identity was of rat origin. Performance qualifications were confirmed by thawing a vial of MCB and monitoring cell viability. Evaluations of copy number, restriction endonuclease spectrum, number of integration sites, integrity of RNA coding sequence and RNA quantification were performed. The heavy chain and light chain copy numbers integrated into the genome were estimated to be 1.13 and 2.14, respectively, by quantitative polymerase chain reaction (Q-PCR). Southern blot evaluation of plasmid integration sites showed that the hybridization patterns of MCB and PSS were comparable. Via Southern blot, the number of integration sites in two cell banks was similarly measured in MCB and WCB, and both were 1. In addition, the chromosomal integration of HK463-25 expression plasmids inserted at a single locus of the mid-centromere chromosome was demonstrated by fluorescence in situ hybridization (FISH) analysis. For MCB, the integrity of the RNA coding sequence was consistent with the reference sequence, and RNA quantification of both heavy and light chains was consistent with the PSS cell pool.

[0540] The MCB was subjected to next generation nucleic acid sequencing (NGS) (Cergentis, Utrecht, Netherlands) using targeted locus amplification (TLA) to confirm that the expressed TG-1101 antibody had the correct amino acid sequence and also to query low-level sequence variants. The MCB was tested for adventitious viruses, mycoplasmas, and microbial substances and met the acceptance criteria established for the release of the MCB, including acceptance criteria for adventitious viruses, mycoplasmas, and microbial substances. The overall MCB test results confirmed the suitability of establishing the MCB.

[0541] Working cell bank batch number G140 / R603 / WCB001

[0542] The first WCB was manufactured at NovaSep (after the acquisition of Henogen). To prepare the WCB, a vial of MCB G071 / MCB / 070208 was thawed and expanded in serum-free medium EM-SF2 P500 H4 in flasks and roller bottles over eleven days. The expanded cell suspension was concentrated by centrifugation and aliquoted into 22 equal parts. Each part was centrifuged and resuspended in freezing medium (90% EM-SF2 P500 H4 + 10% DMSO) to 12.1×10 6 The target cell density was 10 cells / mL. The suspended fractions were then aliquoted into 18 cryovials each, resulting in a total of 396 WCB cryovials. The batch number was named G140 / R603 / WCB001. The cryovials were placed on dry ice and then placed in a cryobox, which was placed in a -80°C freezer for 24 hours. The cryovials were transferred to a liquid nitrogen tank for long-term storage on September 22, 2009. The WCBs are stored in at least two different storage locations, including a small number of vials that were stored at the manufacturer for a shorter period of time. The cell passage number from PSS of the production cell line to WCB is 21.4.

[0543] WCB lot number G140 / R603 / WCB001 was tested including identity testing. Test results confirmed that the WCB identity was of rat origin. Performance qualifications were confirmed by thawing a vial of WCB and monitoring cell viability, doubling time, and IgG productivity. Copy number, restriction endonuclease spectrum, and number of integration sites were evaluated. The heavy chain and light chain copy numbers integrated into the genome were estimated to be 1.2 and 2.5, respectively, by quantitative polymerase chain reaction (Q-PCR). These results are consistent with the results of MCB. Southern blot evaluation of plasmid integration sites demonstrated that the hybridization patterns of WCB and MCB were comparable. The number of integration sites of WCB was measured to be 1 by Southern blot.

[0544] The WCB was subjected to next generation nucleic acid sequencing (NGS) (Cergentis, Utrecht, Netherlands) using targeted locus amplification (TLA) to confirm that the expressed TG-1101 antibody had the correct amino acid sequence and also to query low-level sequence variants. Adventitious virus, mycoplasma and microbial material testing was performed and met all acceptance criteria established for the release of the WCB, including acceptance criteria for adventitious virus, mycoplasma and microbial material. The overall WCB test results confirmed the suitability of establishing the WCB.

[0545] Phenotypic characterization of cell banks

[0546] The MCB and two WCBs generated for the manufacture of TG-1101 were studied for cell line phenotypic stability. Each vial of MCB, WCB batch number G140 / R603 / WCB001 and new WCB batch number 127646-001 were thawed and passaged for about 60 generations. VCD, cell viability and titer samples were collected during each passage. At intervals of about 15 cell generations, cells were cryopreserved as research cell banks (RCBs). After about 60 generations, the vials from each RCB were thawed and amplified for 7 days. On the 7th day, the shake flask was inoculated, cultured under fed-batch conditions, and cultured for 12 days. VCD, cell viability, titer, specific productivity and quality attributes of the fed-batch cultures harvested were sampled every day to assess the stability of each cell bank under its corresponding generation. The comparability of cell growth, titer and product quality was used as a universal basis for determining the phenotypic stability of the cell bank. In addition, a specific productivity result exceeding 70% of the low generation control culture result was used as a specific basis for determining the phenotypic stability of the cell bank. The results of the three cell bank phenotypic stability studies are shown in Table 14 (MCB lot G071 / MCB / 070208), Table 15 (WCB lot G140 / R603 / WCB001) and Table 16 (WCB lot 127646).

[0547] Table 14: Phenotypic stability results of MCB batch G071 / MCB / 070208

[0548]

[0549]

[0550] a Fed-batch culture started after thawing of MCB vials

[0551] Table 15: Phenotypic stability results for WCB batch G140 / R603 / WCB001

[0552]

[0553] a Fed-batch culture started after WCB vials were thawed

[0554] Table 16: Phenotypic stability results for WCB lot 127646

[0555]

[0556] a After thawing of WCB vials to start of production fed-batch culture

[0557] Thawed MCB and WCB used during TG-1101 production at 15,000L were tested for viable cell density and cell viability results to evaluate and confirm cell bank storage stability.

[0558] Upstream Processing and Process Control

[0559] A process flow diagram of the upstream unit operations including operational controls and in-process controls is provided in Table 17. Among other controls, bioburden and endotoxin were measured in batch culture media at the seeding and production bioreactor stages.

[0560] Table 17: Upstream operations and in-process controls

[0561]

[0562]

[0563] Cell culture growth medium (CDM4Mab) and feeds (BalanCD CHOFeed4, glucose feeds and glutamine feeds) were prepared with water for injection (WFI). The medium and feeds were filtered (≤0.2 μm) into sterile containers and stored as needed before use. Cholesterol lipid concentrate was supplemented into the CDM4Mab growth medium during preparation to support cell growth from the inoculum expansion stage to the production bioreactor. In addition, cholesterol lipid concentrate was added to the production bioreactor as a fixed bolus feed additive on process day 0 and day 4. The operation and process controls of the cell culture medium and feeds are described in Table 18.

[0564] Table 18: Cell culture medium and feed preparation and storage controls

[0565]

[0566]

[0567] a Room temperature of SBL: 17-25℃

[0568] b Operating temperature: 37.0℃ (36.5-37.5℃)

[0569] Inoculum expansion

[0570] The inoculum expansion step involves thawing a WCB vial and growing in shake flasks and / or cell bags, increasing the size and volume to provide sufficient cell mass to inoculate the seed bioreactor stage. The step is performed by growing in inoculum expansion growth medium (CDM4Mab). To start the process, thaw a vial of WCB G140 / R603 / WCB001 in pre-warmed hot water in a 37.0°C water bath. Transfer the thawed vial contents to pre-warmed culture medium and dilute to 0.55 x 10 6 The target seeding density is 10 viable cells / mL.

[0571] The cultures were initially placed in 125 mL shake flasks and grown in a shaking incubator at 37.0°C / 5.0% CO2 for 1 day. Every 2 to 3 days, the cultures were expanded to larger shake flasks and / or multiple shake flasks. At each stage, the cultures were seeded at a density of 0.30 x 10 6 The target was 100 viable cells / mL. The final inoculum preparation stage consisted of a 50 L cell bag. After 2 to 3 days of growth, the viable cell density was checked and the culture was further processed to the seed bioreactor stage. The operation and in-process controls for inoculum expansion are described in Table 19.

[0572] Table 19: Inoculum expansion control

[0573]

[0574]

[0575]

[0576] a Shaking platform parabolic radius dependent parameters. Shaking platform parabolic radius: 22mm

[0577] Seed bioreactor

[0578] Before inoculating the production bioreactor, the seed bioreactor stage further increases volume and cell culture biomass. The substratum used in these stages is an inoculum amplification growth medium (CDM4Mab). The seed bioreactor stage is a 120L, 600L and 3000L stainless steel bioreactor. Balance the bioreactor after adding substratum. Correct dissolved oxygen and pH probes before use. The initial bioreactor set point includes temperature, pH, dissolved oxygen and agitation rate. Each bioreactor is inoculated with a cell culture from the previous stage, and the culture is grown for 2 to 3 days. The seed bioreactor operation and control in the process are summarized in Table 20.

[0579] Table 20: Seed bioreactor control

[0580]

[0581]

[0582] a Agitation depends on the volume scale and the bioreactor impeller. Target (NOR / AR) is equivalent to power / volume at SBL

[0583] 20(17-23)W / m 3 .

[0584] Production bioreactors

[0585] The production bioreactor stage is the final cell culture process stage, which further increases the volume and quality of the cell culture for expression of the TG-1101 antibody with acceptable product quality. The basal medium used in this stage is the growth medium (CDM4Mab), and feed additions are performed on specified days or standards during the process. The production bioreactor is a 15,000L stainless steel bioreactor.

[0586] After adding the medium, the production bioreactor was equilibrated. The inoculum expansion medium was added to the initial volume target. The dissolved oxygen and pH probes were calibrated before use. The initial bioreactor set points included temperature, pH, dissolved oxygen, and agitation rate. The bioreactor was heated at 0.5 x 10 6The cell culture from the N-1 seed bioreactor was inoculated with a viable cell density target of 100 viable cells / mL. During the production bioreactor process, pH was controlled as needed using base addition and CO2 sparging. Dissolved oxygen was controlled as needed using oxygen and air sparging. Antifoam was added as needed to alleviate foaming issues. Offline pH, pCO2, pO2, osmotic pressure and metabolites as well as viable cell density (VCD) and viability were monitored daily. BalanCD CHO Feed4 was added at the specified volume (4.0% of the initial bioreactor working volume) on days 3, 5, 7, and 9. When the daily bioreactor sample measurement was <3.00 g / L on the specified process day, the calculated volume of glucose solution (to 4.00 g / L) was fed. A fixed volume of glutamine solution (3.0% of the initial bioreactor working volume) was fed by bolus on day 3, and when the daily bioreactor sample measurement was <3.00 mM on the specified process day, the calculated volume (to 4.00 mM) was fed. The production bioreactor is harvested based on culture duration or cell viability criteria, whichever occurs first.

[0587] Untreated bulk samples were removed prior to the clarification unit operation for extraneous material testing as described in Example 4. The production bioreactor operations and in-process controls are described in Table 21 below.

[0588] Table 21: Production bioreactor controls

[0589]

[0590]

[0591] a Temporary changes are allowed.

[0592] b Agitation depends on the volume scale and the bioreactor impeller. Target (NOR) corresponds to power / volume 50 at SBL

[0593] (42-59)W / m 3 AR is equivalent to power / volume 42-70W / m 3 .

[0594] c Feed was supplemented on days 3, 5, 7 and 9.

[0595] d Feed as needed on days 4-7.

[0596] e After adding the glucose / glutamine feed, glucose / glutamine was not measured; values ​​are those used to determine the required glucose

[0597] Target concentrations used in calculations of glucose / glutamine feed amounts.

[0598] f Feed as needed on days 3-12.

[0599] g The amount of antifoam is calculated based on the initial working volume of the production bioreactor. The goal is to add the amount once.

[0600] h When the culture duration reaches its target / NOR, or when the final cell viability drops below its NOR (in whichever comes first)

[0601] Therefore, when the final cell viability is lower than its NOR, or the culture duration is lower than its

[0602] When NOR, harvesting can be carried out.

[0603] Downstream Processing and Process Control

[0604] A process flow diagram of the downstream steps including operational controls and in-process controls is provided in Table 22. In-process controls have been incorporated into the process as shown in Table 22. Among other controls, bioburden and endotoxin are measured during various stages of the downstream process.

[0605] Table 22: Downstream operations and in-process controls

[0606]

[0607]

[0608] a The beginning, middle, and end of a filling operation

[0609] Harvest clarification

[0610] Cell culture supernatant was harvested and clarified from a 15,000 L bioreactor to remove cells and cell debris. Clarification was performed using sequential centrifugation followed by depth filtration.

[0611] The harvest clarification step is operated in a room with a controlled temperature range of 17 to 25°C; the harvest tank container is a jacketed tank that maintains the tank at 2 to 8°C. The centrifuge shot interval is set based on the percentage cell volume filled (PCV) and is adjusted to allow for 80% bowl fill. The centrifuge flow rate is actively controlled; the filtration feed flow is the same as the centrifuge feed flow. Process parameters include centrate back pressure, depth filtration operating pressure, inlet pressure, harvest weight, bioburden, and endotoxins.

[0612] The centrifuge was clarified using a three-stage filtration process by passing through a 0.2 μm filter (Millistak A1HC POD depth filter, 1.2 / 0.5 μm filter and 0.45 / 0.22 μm sterilizing grade filter) as needed. Before use, the filter was rinsed with WFI and then equilibrated. The centrifuge was pumped through the filter and monitored to ensure acceptable back pressure. The contents of the filter were evacuated using air and then rinsed with buffer. The clarified harvest was stored at 2 to 8°C for ≤11 days.

[0613] Protein A Column Chromatography (ProA)

[0614] Protein A column chromatography was performed using MabSuRe Select resin (Cytiva) in bind / elute mode. This step provided capture and purification of TG-1101, reduced process impurities such as cell culture components, HCPs, and residual DNA, and provided viral safety.

[0615] The HETP performance of packed column was evaluated using sodium acetate / benzyl alcohol buffer. During manufacture, all column operations were carried out at 13 to 25 ° C. Before loading, the column was disinfected with 0.5M sodium hydroxide and rinsed with WFI. Balance column with equilibration buffer (25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1). The harvest clarified was mixed for a short time, then loaded onto the column using a maximum of 36g TG-1101 / L resin load, and washed with Wash 1Buffer (equilibration buffer), then washed for the second time with high salt Wash 2Buffer (25mM Tris, 1.2M NaCl, 5mM EDTA, pH 7.1), and then washed additionally with Wash 3Buffer (equilibrium buffer). The elution peak was collected by A280, and the bound TG-1101 was eluted with elution buffer (25mM sodium citrate, pH 3.6) at 200 to 220cm / h, not exceeding 2.4 column volumes. The eluate was collected in a tank containing neutralization buffer (2.0M Tris, pH 7.5), filtered through a 0.2μm filter, and then transferred to a different tank. The protein A column was disinfected with 0.5M sodium hydroxide. Up to three cycles can be run per batch; if the protein A process requires multiple cycles, the column is re-equilibrated with equilibrium buffer for the next cycle. After disinfection, the protein A column was neutralized with equilibrium buffer and stored at 13 to 25°C in 200mM sodium acetate, 2% benzyl alcohol, pH 5.0. The combined (if more than one cycle), neutralized eluates were diluted with 5 mM sodium phosphate, pH 7.2, to a concentration of ≤10 g / L and stored at 13 to 25 °C for ≤24 hours or at 2 to 8 °C for ≤11 days.

[0616] Solvent Detergent Viral Inactivation (SDVI)

[0617] The protein A capture chromatography step is followed by a solvent detergent virus inactivation (SDVI) step to inactivate potential viral material. In the current validated manufacturing process, the protein A elution pool is diluted and treated with 3.5% (v / v) TnBP, 12% (w / v) polysorbate 80, and maintained at 24.0 to 26.0°C for at least 120 minutes while mixing. The SDVI pool is filtered with a 0.2 μm filter before being transferred to a different tank, diluted to 50 mOsm / kg with 5 mM sodium phosphate (pH 7.2) in the tank, and the pH is adjusted to 7.2 as needed. After pH adjustment, the pool is maintained at 13 to 25°C for ≤30 hours before entering the CEX column.

[0618] Cation Exchange Chromatography (CEX)

[0619] Cation exchange column chromatography was performed using SP Sepharose Fast Flow (Cytiva) in bind / elute mode. This step provided further purification of TG-1101, removing residual process impurities (HCP, DNA, residual polysorbate 80, and TnBP).

[0620] The HETP performance of the packed column was evaluated using a buffer containing sodium acetate / benzyl alcohol. During manufacturing, all column operations were performed at 13 to 25°C. In the currently validated manufacturing process, the column was disinfected with 0.5M sodium hydroxide and rinsed with WFI before loading. The column was balanced with an equilibrium buffer (20mM sodium phosphate, pH 7.2). The virus inactivation / dilution solution was loaded onto the column with a maximum of 65g / L resin load. The column was washed with Wash 1Buffer (equilibrium buffer) and then washed a second time with Wash 2Buffer (equilibrium buffer in reverse). The bound TG-1101 was eluted using 20mM sodium phosphate, 150mM NaCl, pH 7.2, and the elution peak was collected according to A280 monitoring. The eluate was filtered (0.2 μm) and stored at 13 to 25°C for ≤72 hours or stored at 2 to 8°C for ≤11 days. After elution, the column was stripped with 2M NaCl and then disinfected with 0.5M sodium hydroxide. One cycle was allowed per batch. Upon completion, the columns were sanitized (0.5 M sodium hydroxide) and stored in storage buffer (200 mM sodium acetate, 2% benzyl alcohol, pH 5.0).

[0621] Cation Exchange Membrane Chromatography (AEX)

[0622] Anion exchange membrane chromatography was performed in flow-through mode using a Mustang Q (Pall Corporation) membrane absorber (MA) filter. This step provides further purification of TG-1101; the product flows through the membrane, while the remaining impurities (DNA, HCP and viruses) are retained on the membrane. The membrane is disposable (i.e., each individual membrane cannot be reused) and multiple membrane capsules can be used per batch at appropriate documentation levels.

[0623] In order to perform this step, in the currently verified manufacturing process, the eluent from the cation exchange column chromatography step is diluted with 20mM sodium phosphate (pH 8.0), and then the pH is adjusted to 8.0. Determine the concentration and calculate the number of cycles based on protein concentration so that loading is 200 to 700g TG-1101 / L membrane load. The membrane is disinfected with 0.5M sodium hydroxide, rinsed with 2M NaCl, then rinsed with WFI, and then balanced with equilibrium buffer (20mM sodium phosphate, 75mM NaCl, pH 8.0) to prepare for loading. After loading, rinse the membrane with 20mM sodium phosphate, 75mM NaCl, pH 8.0 to maximize the recovery. The product containing effluent collected in all cycles is filtered (0.5 / 0.2 μm), diluted to ≤6g / L with 75mM sodium citrate, 312mM NaCl, pH 6.0, and the pH is adjusted to 6.8. The conditioned AEX pool was stored at 13 to 25°C for ≤72 hours or at 2 to 8°C for ≤11 days.

[0624] Virus Filtration (VF)

[0625] Virus filtration was performed through a Viresolve pre-filter in series with a Viresolve Pro virus filter (Millipore Sigma) at an operating temperature target range of 13 to 25°C. The process used enough filters to meet the loading limit. This step was designed to remove potential viruses, including small viruses such as parvovirus.

[0626] To perform the filtration, the filters were placed in series and rinsed with WFI, tested for integrity, and then sterilized with 0.5M sodium hydroxide. They were then rinsed with an equilibration buffer (25mM sodium citrate, 154mM NaCl, pH 6.5). The filtered Mustang Q membrane effluent was processed through a virus reduction filter.

[0627] Determine protein concentration and use to confirm membrane loading ≤ 600 g / m 2 After loading, the membrane was rinsed with equilibration buffer and subjected to a post-use integrity test. Viral filtrates were stored at 13 to 25 °C for ≤72 hours or at 2 to 8 °C for ≤11 days.

[0628] Ultrafiltration / diafiltration (UFDF) control

[0629] The UFDF step is used to concentrate the viral filtrate and buffer exchange into the diafiltration buffer at an operating temperature target range of 13 to 25°C. The process uses a tangential flow filter with a molecular weight cutoff of 30 kDa. The setup includes enough filters to meet ≤250 g / m 2 To perform this operation, the membrane was sanitized with 0.5 M NaOH, rinsed with WFI, and flushed / equilibrated with diafiltration buffer before the start of the unit operation.

[0630] The unit operation includes an initial ultrafiltration (UF1) step, in which TG-1101 is first concentrated to a target of 39 mg / mL. It is then diafiltered (DF) into a diafiltration buffer (25 mM sodium citrate 154 mM NaCl, pH 6.5) with 8 diafiltration volumes (upper limit ≤ 9.2 diafiltration volumes). The ultrafiltration system is rinsed with diafiltration buffer to maximize recovery; the rinse is transferred to a preparation container and merged with the UF / DF pool. The membrane is rinsed with WFI, cleaned with 0.5 M NaOH, 250 ppm sodium hypochlorite, rinsed with WFI, and stored in 0.1 M sodium hydroxide. The diluted UFDF pool is stored at 15 to 25 ° C for ≤ 18 hours.

[0631] Formulating, filtering, and filling

[0632] The formulation step involves the addition of concentrated polysorbate 80 in a formulation buffer to obtain a final drug substance formulation at an operating temperature target range of 17 to 25°C. This step is performed by adding a stabilization buffer (25 mM sodium citrate, 154 mM NaCl, 10 g / L polysorbate 80, pH 6.5). After the addition of the stabilization buffer, the pool is diluted to a target of 23.5 to 26.5 mg / mL with 25 mM sodium citrate, 154 mM sodium chloride, 700 mg / L polysorbate 80, pH 6.5, resulting in a ready-to-fill drug substance in a formulation buffer of 25 mM sodium citrate, 154 mM NaCl, 0.07% polysorbate 80, pH 6.5.

[0633] Transfer formulated bulk API and 0.2 μm filter into 6 L sealed single-use system FFT bags were filled to a target fill volume of 5.50 L. After filling, the formulated bulk drug substance was frozen at ≤ -60 °C for >17 hours and stored at ≤ -35 ​​°C.

[0634] Example 4: Detection of viruses and foreign substances in 15,000 commercial-scale production of TG-1101 in YB2 / 0 rat hybridoma cells

[0635] The adventitious and viral safety of TG-1101 was ensured through several strategies in a multi-pronged approach. These strategies included preventing the introduction of adventitious agents into the manufacturing process, removing them with dedicated process steps, and testing them at appropriate points in the process. In addition, testing for adventitious viruses was performed to confirm the absence of adventitious viruses during cell line / cell bank development and manufacturing. Virus clearance studies confirmed the ability of the manufacturing process to clear viruses.

[0636] During the manufacture of TG-1101, a comprehensive viral safety strategy consisted of the following approaches and activities:

[0637] Raw material procurement

[0638] No raw materials of animal or biological origin are used directly in the upstream manufacturing process of TG-1101. For example, the cell culture media and feeds used during manufacturing, CDM4Mab Cell Growth Medium and BalanCD-CHO Feed 4 are proprietary commercially available cell culture media. Both of these media are animal component-free, and no ingredients are identified as being in contact with raw materials of animal origin in their manufacture. During the downstream manufacturing process, the protein A chromatography resin is not derived from materials of animal origin and the cation chromatography resin does not contain any raw materials from animal origin.

[0639] Testing of production cell lines during development and subsequently derived cell banks (MCB, WCB and EPCB)

[0640] The cell bank system of TG-1101 is a two-layer system made of a master cell bank (MCB) and several working cell banks (WCB). Cell bank (i.e. MCB and WCB) foreign substance testing includes mycoplasma, microorganisms, non-endogenous viruses and exogenous viruses. The final production cell bank (EPCB) derived from the WCB is additionally tested for foreign substances (microbial and viral).

[0641] Evaluation of foreign matter during the manufacturing of TG-1101 API

[0642] Specifically, routine mycoplasma, microbial and viral adventitious testing of unprocessed bulk harvest (from production bioreactors) was performed, as well as in-process bioburden and endotoxin testing throughout the manufacturing process.

[0643] Virus clearance studies

[0644] Virus clearance testing in a typical scale-down model illustrates the ability of the manufacturing process to inactivate and / or eliminate model viruses. Virus clearance studies were performed using four model viruses, including xenotropic murine leukemia virus (XMuLV), a specific model virus of the retroviral family. Three additional non-specific model viruses were selected to provide a range of viruses and sizes and resistance to chemical inactivation. Minute Virus of Mice (MVM), a model of parvovirus, was selected as a virus representing a small size and high resistance to chemical inactivation, and is also a potential virus for contamination of mammalian cell lines. Virus clearance of pseudorabies virus (PRV) and reovirus type 3 (Reo 3) was also tested.

[0645] The TG-1101 manufacturing process includes specific, orthogonal, dedicated viral inactivation / removal steps, including a solvent / detergent inactivation step and a viral filtration step. The manufacturing process includes several chromatographic steps that also aid in viral inactivation / removal during the manufacturing process. Collectively, the extraneous material controls in the manufacturing process provide a high degree of assurance that the TG-1101 drug substance is adequately safe with respect to extraneous materials.

[0646] Example 5: Effect of pH after conversion on process performance and product quality of TG-1101 upstream manufacturing process

[0647] background

[0648] Throughout the development of the upstream portion of the commercial TG-1101 manufacturing process ("Process C"), a large number of experiments and data analysis were performed. Culture pH was identified as an important parameter that can affect process performance and product quality. pH control of the commercial manufacturing process (15,000L) ("Process C2", commercial process) run at Samsung Biologics (SBL) follows the control strategy shown in Table 23.

[0649] Table 23: pH control strategy for process C2

[0650]

[0651] a AR = Acceptable Range

[0652] Based on both the 15kL and 20kL GMP manufacturing results, as well as the scaled-down model (10L) results, the cumulative cell exposure to pH after the pH shift on day 3 (72 hours) was identified as being particularly important to process performance. The latter calculated parameter is defined as the “integrated pH2 difference” and can be conceptually viewed as Fig.21 The shaded portion is shown in FIG.

[0653] Materials and methods

[0654] Cell culture

[0655] The mammalian cell expression system used is the TG-1101YB2 / 0 rat hybridoma cell line amplified from the working cell bank lot #G140 / R603 / WCB001 or lot #127646 as described above. After the frozen vial of the cell from any WCB is thawed, the individual cell expansion is carried out in the shaking flask container and disposable cell bag bioreactor of increasing volume. The cell is expanded for multiple passages in CDM4Mab (Cytiva) cell culture medium supplemented to 1X cholesterol level using 1000X cholesterol (Thermo). After the further cell expansion in the seed bioreactor culture, when enough cell inoculum is generated, the production culture is operated in fed-batch mode. During the fed-batch production culture, BalanCD CHO Feed4 (Fuji) is added with fixed volume at the 3rd, 5th, 7th and 9th day of the process. In addition, the dedicated glutamine feed supplement is added at the 3rd day. Glutamine and glucose feed supplements are added according to pre-established standards. In general, cell culture process conditions were similar in each different culture analyzed, except for normal or intentionally introduced culture pH drifts.Cell culture growth medium, feed medium and feed solutions were prepared in a manner consistent with the spirit of GMP manufacturing.

[0656] In-process monitoring is performed throughout the duration of the production bioreactor culture. Included among these samples is the daily measurement of viable cell density (VCD) and cell viability by an automated cell counter (ViCell XR, Beckman Coulter). Nova Flex2 (Nova Biomedical) instruments are used for metabolite measurements (glucose, lactate, glutamine, ammonia). Nova pHOx (Nova Biomedical) and / or Siemens blood gas analyzers are used to measure offline pH, pO2, and pCO2 as needed. Nova Flex 2 (Nova Biomedical) and Advanced Instruments osmometers are used to measure osmotic pressure. For titer and product quality measurements, cells are removed from the culture, clarified by centrifugation, and kept in storage until ready for analysis.

[0657] Protein analysis and functional assays

[0658] N-Glycans

[0659] N-linked glycans are cut from the product by enzymatic deglycosylation using PNGase F and labeled with 2-aminobenzamide (2-AB), a fluorescent compound. Labeled glycans are resolved using a hydrophilic interaction partitioning mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360nm and emission at 428nm). Blanks, glycans from reference standards, and glycan standards (human IgG N-linked glycan library) are also injected to assess system suitability. Peak identification from the resulting test sample chromatogram is based on retention time and is identified relative to the peak in the glycan standards confirmed by mass spectrometry.

[0660] Results and Conclusions

[0661] The post-transition pH (pH2) followed a trajectory that was generally consistent in its trend. That is, around day 7-8 of the production bioreactor culture, lactate began to increase to appreciable amounts, whereby the culture pH dropped to the lower end of its control range. The rate and timing of this drop in pH varied between the Process C GMP manufacturing batches and the 10 L scale-down model cultures ( Fig. 22 As a result, the cumulative cell exposure to pH in each of these cultures was different, and as a result, the integrated pH2 difference was also different. The integrated pH2 difference has an inverse relationship with the cumulative number of cells generated during the production bioreactor, in that the lower the integrated pH2 difference, the higher the integrated viable cell density (IVCD) ( Fig.23 The integrated pH2 difference also had an inverse relationship with the titer results at harvest due to higher cell numbers ( Fig.24 ). Due to the constitutive secretion of higher amounts of TG-1101 from the cells, the % fucosylation was generally lower in those cultures showing lower integrated pH2 differences ( Fig.25 ). These results indicate that if the goal is to reduce fucosylation %, such as by switching the TG-1101 manufacturing process from process A / B to process C, this can be achieved by introducing a process change that increases TG-1101 productivity. Minimizing the integrated pH2 difference is one means to achieve this.

[0662] The post-conversion pH was directly controlled in the commercial TG-1101 manufacturing process using a pH control strategy that prevented the fucosylation % from exceeding the range of the drug substance (DS) release specification. When the post-conversion pH was controlled within its control range at commercial manufacturing scale, the fucosylation % results were observed to be relatively stable in the range of 22-37%.

[0663] Example 6: Unique glycosylation characteristics of anti-CD20 antibodies produced in YB2 / 0 rat hybridoma cells at 15,000 L commercial scale

[0664] Using the manufacturing method disclosed herein (e.g., such as described in Example 3), anti-CD20 antibodies are produced by source antibody TG-1101 at a scale of 15,000L. It was found that anti-CD20 antibodies contain unique glycosylation profiles. Without being bound by theory, the relative distribution of various N-glycans or individual sugar residues present in these N-glycans can determine the biological and clinical properties of the anti-CD20 antibody compositions provided herein. See, co-pending (and co-owned) U.S. Provisional Application No. 63 / 347,852, entitled “ANTI-CD20ANTIBODY COMPOSITIONS”, filed on June 1, 2022, U.S. Provisional Application Serial No. 63 / 421,078, filed on October 31, 2022, and U.S. Provisional Application Serial No. 63 / 445,082, filed on February 13, 2023, the entire contents of which are incorporated herein by reference. The anti-CD20 antibody compositions described, as well as those described herein, can be used to treat cancer (eg, hematological cancers) and autoimmune disorders (eg, RMS).

[0665] The glycosylation profile of the anti-CD20 antibody sample was determined by measuring fluorescently labeled N-glycans (fluorescent label is 2-aminobenzamide) which were cleaved from the anti-CD20 antibody protease using PNGase F. The labeled glycans were resolved using a hydrophilic interaction column equipped with the HPLC-MS / MS. The glycans flowed through a fluorescence detector after separation. Peak identification from the test sample chromatogram was based on retention time and relative to peaks in a glycan standard that had been confirmed by mass spectrometry. The relative percentage of each N-glycan was calculated based on the N-glycan peak area divided by the total peak area of ​​all N-glycans. The glycosylation profile is shown in Fig.26 middle.

[0666] The glycosylation profile of anti-CD20 antibodies was evaluated by intact mass analysis (LC-MS) under non-reducing conditions. During the chromatography step using SEC and a mobile phase containing TFA, acetonitrile and water, the sample of anti-CD20 antibodies was first exchanged into a suitable buffer for MS. The sample was then introduced into ESI-QTOF for intact mass analysis. The mass spectrum was deconvoluted and peaks were assigned based on mass. The relative abundance of each anti-CD20 antibody provided herein containing N-glycans was calculated by taking the abundance of N-glycans and dividing by the total abundance of all identified peaks. The results are provided in Table 24 below.

[0667] Table 24: Intact molecular weight of anti-CD20 antibody samples by LC-MS

[0668]

[0669]

[0670] Abbreviations: ND = not detected.

[0671] The anti-CD20 antibodies provided herein can be described by any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all of the N-glycans or individual sugar residues described in the following subsections.

[0672] In some aspects, the anti-CD20 antibodies provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0673] (a) 0.3% to 2% GO-GN;

[0674] (b) 0.1% to 2% GOF-GN;

[0675] (c) 0.1% to 1% G1-GN;

[0676] (d) 5% to 20% GOB;

[0677] (e) 5% to 30% GOF;

[0678] (f) 0.1% to 1.5% Man5;

[0679] (g) 1% to 15% GOFB;

[0680] (h) 1% to 13% G1;

[0681] (i) 0.5% to 10% G1';

[0682] (j) 0.5% to 6% G1B;

[0683] (k) 0.5% to 12% G1F;

[0684] (l) 0.1% to 3% G1F';

[0685] (m) 0.1% to 3% G1FB;

[0686] (n) 0.1% to 2% G2; and

[0687] (o) 0.1% to 2% G2F.

[0688] In more specific aspects, the anti-CD20 antibodies provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0689] (a) 0.8% to 1.1% GO-GN;

[0690] (b) 0.5% to 1.1% GOF-GN;

[0691] (c) 0.3% to 0.6% G1-GN;

[0692] (d) 9.5% to 14.1% GOB;

[0693] (e) 12.8% to 19.7% GOF;

[0694] (f) 0.4% to 0.7% Man5;

[0695] (g) 5.1% to 7.0% GOFB;

[0696] (h) 5.7% to 6.4% G1;

[0697] (i) 2.7% to 3.3% G1';

[0698] (j) 1.4% to 2.0% G1B;

[0699] (k) 2.6% to 4.2% G1F;

[0700] (l) 1.1% to 1.6% G1F';

[0701] (m) 1.1% to 1.8% G1FB;

[0702] (n) 0.5% to 0.7% G2; and

[0703] (o) 0.3% to 0.5% G2F.

[0704] In even more specific aspects, the anti-CD20 antibodies provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0705] (a) 0.9% G0-GN;

[0706] (b) 0.8% GOF-GN;

[0707] (c) 0.5% G1-GN;

[0708] (d) 10.9% G0B;

[0709] (e) 17.0% GOF;

[0710] (f) 0.6% Man5;

[0711] (g) 6.0% G0FB;

[0712] (h) 6.1% G1;

[0713] (i) 2.9% G1';

[0714] (j) 1.6% G1B;

[0715] (k) 3.2% G1F;

[0716] (l) 1.3% G1F';

[0717] (m) 1.3 G1FB;

[0718] (n) 0.5% G2; and

[0719] (o) 0.3% G2F.

[0720] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising a relative abundance of from about 0.3% to about 2% GO-GN, from about 0.8% to about 1.1% GO-GN, or about 0.9% GO-GN. In certain embodiments, the anti-CD20 antibodies comprise an N-glycan profile comprising a relative abundance of from about 0.1% to about 2% GOF-GN, from about 0.5% to about 1.1% GOF-GN, or about 0.8% GOF-GN. In certain embodiments, the anti-CD20 antibodies comprise an N-glycan profile comprising a relative abundance of from about 0.1% to about 1% G1-GN, from about 0.3% to about 0.6% G1-GN, or about 0.5% G1-GN. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising the following relative abundances: from about 5% to about 20% GOB, from about 5% to about 15% GOB, from about 9.5% to about 14.1% GOB, about 10.9% GOB, or about 10% GOB. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising the following relative abundances: from about 5% to about 30% GOB, from about 12.8% to about 19.7% GOB, or about 17.0% GOB. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising the following relative abundances: from about 0.1% to about 1.5% Man5, from about 0.4% to about 0.7% Man5, or about 0.6% Man5. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising a relative abundance of from about 1% to about 15% GOFB, from about 5.1% to about 7.0% GOFB, or about 6.0% GOFB. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising a relative abundance of from about 1% to about 13% G1, from about 5.7% to about 6.4% G1, or about 6.1% G1. In certain embodiments, the anti-CD20 antibody protein comprises an N-glycan profile comprising a relative abundance of from about 0.5% to about 10% G1', from about 2.7% to about 3.3% G1', or about 2.9% G1'. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising a relative abundance of from about 0.5% to about 6% G1B, from about 1.4% to about 2.0% G1B, or about 1.6% G1B. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile comprising relative abundances of from about 0.5% to about 12% G1F, from about 2.6% to about 4.2% G1F, or about 3.2% G1F.In certain embodiments, the anti-CD20 antibodies comprise an N-glycan profile comprising a relative abundance of from about 0.1% to about 3% G1F', from about 1.1% to about 1.6% G1F', or about 1.3% G1F'. In certain embodiments, the anti-CD20 antibodies comprise an N-glycan profile comprising a relative abundance of from about 0.1% to about 3% G1FB, from about 1.1% to about 1.8% G1FB, or about 1.3G1FB. In certain embodiments, the population of anti-CD20 antibodies comprises an N-glycan profile comprising a relative abundance of from about 0.1% to about 2% G2, from about 0.5% to about 0.7% G2, or about 0.5% G2. In certain embodiments, the anti-CD20 antibodies comprise an N-glycan profile comprising a relative abundance of from about 0.1% to about 2% G2F, from about 0.3% to about 0.5% G2F, or about 0.3% G2F.

[0721] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising the following relative abundances: from about 0.3% to about 2% GO-GN, from about 0.1% to about 2% GOF-GN, from about 0.1% to about 1% G1-GN, from about 5% to about 20% G0B, from about 5% to about 30% GOF, from about 0.1% to about 1.5% Man5, from about 1% to about 15% GOFB, from about 1% to about 13% G1, from about 0.5% to about 10% G1', from about 0.5% to about 6% G1B, from about 0.5% to about 12% G1F, from about 0.1% to about 3% G1F', from about 0.1% to about 3% G1FB, from about 0.1% to about 2% G2, and from about 0.1% to about 2% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0722] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising the following relative abundances: from about 0.8% to about 1.1% GO-GN, from about 0.5% to about 1.1% GOF-GN, from about 0.3% to about 0.6% G1-GN, from about 9.5% to about 14.1% G0B, from about 12.8% to about 19.7% G0F, from about 0.4% to about 0.7% Man5, from about 5.1% to about 7.0% GOFB, from about 5.7% to about 6.4% G1, from about 2.7% to about 3.3% G1', from about 1.4% to about 2.0% G1B, from about 2.6% to about 4.2% G1F, from about 1.1% to about 1.6% G1F', from about 1.1% to about 1.8% G1FB, from about 0.5% to about 0.7% G2, and from about 0.3% to about 0.5% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0723] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising the following relative abundances: about 0.9% GO-GN, about 0.8% GOF-GN, about 0.5% G1-GN, about 10.9% G0B, about 17.0% G0F, about 0.6% Man5, about 6.0% GOFB, about 6.1% G1, about 2.9% G1', about 1.6% G1B, about 3.2% G1F, about 1.3% G1F', about 1.3G1FB, about 0.5% G2, and about 0.3% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0724] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising G1 and G0 N-glycans in a relative abundance ratio of about 0.1 to about 0.15. In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile comprising G1F and G1 N-glycans in a relative abundance ratio of about 0.5 to about 0.9.

[0725] The present invention is not limited in scope by the specific embodiments described herein. In fact, from the above description and the accompanying drawings, various modifications of the present invention other than those described will become apparent to those skilled in the art. Such modifications are intended to fall within the scope of the appended claims.

[0726] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein.

Claims

1. A method for producing at least 10,000 L of an antibody protein in a rat hybridoma cell, the method comprising culturing the rat hybridoma cell in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cell comprises an expression vector comprising a polynucleotide encoding the antibody protein.

2. The method of claim 1, wherein the culture pH is about 6.5 to about 7.

0.

3. The method according to claim 2, wherein the culture pH of about 6.5 to about 7.0 is set on culture day 2 of the cell culture.

4. The method according to claim 2, wherein the culture pH of about 6.5 to about 7.0 is set on culture day 3 of the cell culture.

5. The method of claim 1, wherein the culture pH is about 7.0 to about 7.

55. 6 . The method according to claim 5 , wherein the culture pH of about 7.0 to about 7.55 is set on culture day 0 to day 3 of the cell culture.

7. The method according to claim 5 or 6, wherein the culture pH is lowered to about 6.5 to about 7.0 on day 2 or day 3 of cell culture. The method according to claim 7 , wherein the culture pH is lowered on day 3 of cell culture.

9. The method according to claim 7 or 8, wherein the culture pH is maintained at about 6.5 to about 7.0 from culture day 3 of the cell culture until harvest.

10. The method of claim 8 or 9, wherein the cumulative culture time and the cumulative decrease in pH (integrated pH2 difference) allowed to drop below the fixed pH set point after the pH is reduced on day 3 are smaller relative to cell cultures with a larger integrated pH2 difference.

11. The method of claim 10, wherein the fixed pH set point is pH 6.

91.

12. The method of claim 10 or 11, wherein a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and a higher titer at harvest.

13. The method according to any one of claims 10 to 12, wherein a lower integrated pH2 difference further results in a lower fucosylation percentage.

14. The method according to any one of claims 1 to 13, wherein the rat hybridoma cells expressing the antibody protein are cultured in a chemically defined medium free of animal-derived components (ADCF).

15. The method according to any one of claims 1 to 14, wherein the harvest titer of the antibody protein is increased and / or the fucosylation of the antibody protein is reduced when the culture pH is 6.6 to 6.96, relative to cell culture under the same culture conditions except that the culture pH is 6.60 to 6.

80.

16. The method of any one of claims 1 to 15, further comprising controlling the culture pCO2 level to less than about 300 mmHg.

17. The method of claim 16, wherein a pCO2 level of less than about 300 mmHg is promoted by supplementing the cell culture with additional buffer, increasing the air sparging rate, increasing the dissolved oxygen (DO) set point, and / or reducing the agitation rate.

18. The method according to any one of claims 1 to 17, further comprising an initial temperature set point of about 37°C, wherein the initial temperature set point is set at culture day 0 to culture day 1.

19. The method of claim 18, further comprising a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3.

20. The method of claim 19, wherein the end of culture day 1 is 17 to 33 hours after initiation of cell culture.

21. The method according to any one of claims 18 to 20, further comprising a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest.

22. The method of claim 21, wherein the third temperature set point is 32.5°C.

23. The method according to any one of claims 1 to 22, wherein the cell culture comprises the following culture conditions: i) an initial temperature set point of about 37°C, wherein the initial temperature set point is set from culture day 0 to culture day 1; a second temperature set point of about 35°C, wherein the second temperature set point is set from the end of culture day 1 to culture day 3; and a third temperature set point of about 32.5°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

24. The method of any one of claims 1 to 23, wherein the yield of the antibody protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150% relative to an antibody protein produced by a culture process that does not employ the culture conditions of claim 23.

25. The method according to any one of claims 1 to 24, further comprising harvesting the antibody protein produced by rat hybridoma cells.

26. The method of claim 25, further comprising purifying the antibody protein by affinity chromatography and / or ion exchange chromatography, optionally wherein the affinity chromatography comprises Protein A purification.

27. The method of claim 26, wherein the purified antibody protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation.

28. The method of claim 26 or 27, wherein the quality of the purified antibody protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis.

29. The method of any one of claims 1 to 28, wherein the antibody protein is a monoclonal antibody.

30. The method of claim 29, wherein the monoclonal antibody is an anti-CD20 antibody.

31. The method of claim 29 or 30, wherein the monoclonal antibody is subjected to a CD20, FcγRIIIa-158V and / or C1q binding assay.

32. The method of claim 31 , wherein the monoclonal antibody has a relative potency of 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard.

33. The method of claim 32, wherein CD20 binding is determined by binding of an anti-CD20 antibody to the CD20 expressing human mantle cell lymphoma cell line Jeko-1.

34. The method of any one of claims 31-33, wherein the percentage of FcγRIIIa-158V binding relative to a commercial reference standard used in the binding assay is about 82% to about 130%.

35. The method of claim 34, wherein the percentage of FcγRIIIa-158V binding is determined by surface plasmon resonance (SPR).

36. The method of any one of claims 31-35, wherein the monoclonal antibody has a relative potency of 86% to 117% in a CIq binding assay as measured by ELISA compared to a commercial reference standard.

37. The method of claim 36, wherein the monoclonal antibody has a relative potency in a CIq binding assay of 88% to 113% as measured by ELISA compared to a commercial reference standard.

38. The method of any one of claims 29-37, wherein the monoclonal antibody has a relative potency of 74% to 127% in a cell-based complement dependent cytotoxicity (CDC) assay compared to a commercial reference standard.

39. The method of any one of claims 29-38, wherein the monoclonal antibody produced has a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity relative to a monoclonal antibody produced by a culture process that does not employ the culture conditions of claim 23.

40. The method of claim 38, wherein the monoclonal antibody has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard.

41. The method of claim 40, wherein the monoclonal antibody has a relative potency of about 117% in a cell-based ADCC assay compared to a commercial reference standard.

42. The method of any one of claims 30-41, wherein the monoclonal antibody comprises: a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

43. The method of claim 42, wherein the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7 and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

8.

44. The method of claim 42 or 43, wherein the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:

8.

45. The method of claim 42 or 43, wherein the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:

9.

46. ​​The method of any one of claims 42-45, wherein the monoclonal antibody comprises a deletion of up to 5 N-terminal residues.

47. The method of any one of claims 42-45, wherein the monoclonal antibody comprises a deletion of up to 10 N-terminal sequences.

48. The method of any one of claims 1-47, wherein the cell cultivation is performed in a bioreactor.

49. The method of claim 48, wherein the bioreactor is a commercial scale bioreactor.

50. The method of claim 49, wherein the commercial scale bioreactor is a 10,000L, 15,000L, 20,000L or 25,000L bioreactor.

51. The method of claim 50, wherein the commercial scale bioreactor is a 15,000 L bioreactor.

52. The method of any one of claims 1-51, wherein the rat hybridoma cell is a YB2 / 0 rat hybridoma cell.

53. A method for producing antibody proteins in culture of rat hybridoma cells on a commercial scale, the method comprising the steps of: a) preparing and thawing a working rat hybridoma cell bank of the antibody protein of interest; b) Expand the size and volume of cultures of rat hybridoma cells from a cell bank through a series of shake flasks (125 mL, 500 mL, 3 L, 3x3 L shake flasks, and 50 L cell bags) with a target seeding density of at least 0.30 x 10 6 viable cells / mL; c) processing the cell culture through a series of seed bioreactors (120 L, 600 L, and 3,000 L) to further increase the volume and cell culture quality; d) inoculating the cell culture from the 3,000 L seed bioreactor into the commercial scale production bioreactor; e) harvesting cell culture supernatant from the commercial scale production bioreactor; f) clarifying the recovered cells by sequential centrifugation followed by depth filtration; g) purifying the antibody protein by protein A capture column chromatography; and h) Inactivation of viral material by solvent detergent viral inactivation (SDVI).

54. The method of claim 53, wherein the commercial scale production bioreactor is operated in fed-batch mode.

55. The method of claim 53 or 54, wherein a drilled hole (10) gas injector having an orifice diameter of 4.0 mm is used in the commercial scale production bioreactor.

56. The method of any one of claims 53-55, further comprising purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX).

57. The method of any one of claims 53-56, further comprising viral filtration (VF) to remove potential viruses.

58. The method of any one of claims 53-57, further comprising ultrafiltration / diafiltration (UFDF).

59. The method of any one of claims 53-58, further comprising preparing a bulk drug substance formulation comprising the antibody protein, comprising adding polysorbate 80 to a formulation buffer to prepare the bulk drug substance formulation.

60. The method of claim 59, further comprising subjecting the bulk drug substance formulation to 0.2 μm filtration.

61. The method of claim 59 or 60, further comprising filling a 6 L bag to a target fill volume of 5.50 L of the bulk drug substance formulation, and storing the bulk drug substance formulation at ≤ -35°C.

62. The method of any one of claims 59-61, wherein the antibody protein in the bulk drug substance formulation is formulated into a pharmaceutically acceptable formulation.

63. The method of any one of claims 59-62, further comprising testing unprocessed bulk harvest from the commercial scale production bioreactor for microbial and viral adventitious materials.

64. The method of claim 63, further comprising removing and / or inactivating the microbial and viral extraneous matter from the commercial scale production bioreactor.

65. according to the method described in any one of claims 53-64, wherein said rat hybridoma cell is YB2 / 0 cell.

66. The method of any one of claims 53-65, wherein the antibody protein is a monoclonal antibody, optionally wherein the monoclonal antibody is an anti-CD20 antibody.

67. The method of claim 66, wherein the monoclonal antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

68. The method of claim 67, wherein the monoclonal antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

8.

69. The method of claim 67 or 68, wherein the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:

8.

70. The method of claim 67 or 68, wherein the monoclonal antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:

9.

71. The method of any one of claims 1-70, wherein the antibody protein comprises an N-glycan profile comprising one or both of: i) about 10 to 20% galactosylated polysaccharides; and / or ii) about 20 to 40% fucosylated glycans.

72. The method of claim 71, wherein the N-glycan profile comprises about 10 to 20% galactosylated glycans and about 23% to 36% fucosylated glycans.

73. The method of claim 71 or 72, wherein the N-glycan profile comprises about 23% to about 36% fucosylated glycans.

74. The method of any one of claims 71-73, wherein the N-glycan profile comprises about 16% to about 18% galactosylated glycans, optionally about 17% galactosylated glycans.

75. The method of any one of claims 1-74, wherein the antibody protein comprises an N-glycan profile comprising at least about 10% bisecting N-glycans.

76. The method of claim 75, wherein the N-glycan profile comprises about 12% to about 30% bisecting N-glycans.

77. The method of claim 76, wherein the N-glycan profile comprises about 18% bisecting N-glycans.

78. The method of any one of claims 1-77, wherein the antibody protein comprises an N-glycan profile comprising less than 5% sialylated glycans.

79. The method of claim 78, wherein the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycans.

80. The method of claim 78 or 79, wherein the N-glycan profile comprises undetectable amounts of sialylated glycans.

81. The method of any one of claims 1-80, wherein the antibody protein comprises an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans.

82. The method of claim 81, wherein the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans.

83. The method of claim 82, wherein the N-glycan profile comprises about 0.6% Man5 N-glycans.

84. The method of any one of claims 81-83, wherein Man5 N-glycans are the only high mannose species in the N-glycan profile.

85. The method of any one of claims 1-84, wherein the antibody protein is produced at a commercial scale of about 10,000 L to about 25,000 L.

86. The method of claim 85, wherein the commercial scale is 15,000 L.

87. The method of any one of claims 1-86, wherein the method produces an antibody protein harvest titer of about 0.5 g / L to about 1.5 g / L.

88. The method of claim 87, wherein the harvest titer is about 1.0 g / L to about 1.5 g / L.

89. An antibody protein produced according to the method of any one of claims 1-88.

90. The antibody protein of claim 89, wherein the antibody protein is a monoclonal antibody, optionally wherein the monoclonal antibody is an anti-CD20 antibody.

91. A rat hybridoma master cell bank (MCB) composition comprising antibody proteins having at least two of the following parameters: i) about 11 to about 13 x 10 6 The peak viable cell density was cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a fucosylation percentage of about 30% to about 38%; iv) about 97% to about 99% monomer as determined by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimer as detected by SEC; vi) undetectable levels to about 3% levels of aggregates as detected by SEC; vii) undetectable levels to about 1% levels of fragments as detected by SEC; viii) about 25% to about 30% acidic isoform as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 38% to about 49% major isoform as detected by iCIEF; and / or x) about 20% to about 36% basic isoform as detected by iCIEF.

92. A rat hybridoma working cell bank (WCB) composition comprising antibody proteins having at least two of the following parameters: i) from about 11 to about 28 x 10 6 The peak viable cell density was cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a fucosylation percentage of about 18% to about 40%; iv) about 97% to about 99% monomer as determined by size exclusion chromatography (SEC); v) about 1% to about 2% dimer as detected by SEC; vi) undetectable levels to about 2% levels of aggregates as detected by SEC; vii) undetectable levels to about 1% levels of fragments as detected by SEC; viii) about 19% to about 31% acidic isoform as detected by imaged capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoform as detected by iCIEF; and / or x) about 14% to about 38% basic isoform as detected by iCIEF.

93. The MCB composition of claim 91 or the WCB composition of claim 92, wherein the rat hybridoma cells in the cell bank are YB2 / 0 cells.

94. The MCB or WCB composition of claim 93, wherein the antibody protein is a monoclonal antibody, optionally wherein the monoclonal antibody is an anti-CD20 antibody.

95. The MCB or WCB composition of claim 94, wherein the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

96. The MCB or WCB composition of claim 95, wherein the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO:7; and a light chain having at least 95% identity to the amino acid sequence shown in SEQ ID NO:

8.

97. The MCB or WCB composition of claim 95 or 96, wherein the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:

8.

98. The MCB or WCB composition of claim 95 or 96, wherein the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:

9.

99. A method of producing an antibody protein by using the MCB or WCB composition according to any one of claims 91-98.

100. The method of claim 99, wherein the antibody protein is a monoclonal antibody, optionally wherein the monoclonal antibody is an anti-CD20 antibody.

101. The method of claim 100, wherein the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5; and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

102. The method of claim 101, wherein the anti-CD20 antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; and a light chain that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

8.

103. The method of claim 101 or 102, wherein the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7 and a light chain having the amino acid sequence shown in SEQ ID NO:

8.

104. The method of claim 101 or 102, wherein the anti-CD20 antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:7; and a light chain having the amino acid sequence shown in SEQ ID NO:9.

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