Commercial-scale recombinant protein production in rat hybridoma cells

Abstract

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

Description

Technical Field

[0001] Claims of Priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 347,793, filed Jun. 1, 2022. The entire foregoing is hereby incorporated by reference herein.

[0002] Reference to Electronically Submitted Sequence Listing This application contains a sequence listing that was electronically submitted as an XML file named “50581-0003WO1.XML”. The XML file, created on May 22, 2023, is 19,157 bytes in size. The material in the XML file is hereby incorporated by reference herein in its entirety.

[0003] The present disclosure is in the field of mammalian cell culture for producing recombinant proteins (e.g., monoclonal antibodies). More specifically, the present disclosure is in the field of commercial-scale production (e.g., 10,000 L to 25,000 L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells.

Background Art

[0004] Recombinant proteins (e.g., antibodies) have become increasingly important as therapeutic agents in a wide range of diseases such as cancer, autoimmune, and infectious diseases. See Kaplon, H. et al., MAbs 11:219-238 (2019); Lu, R-M. et al., Journal of Biomedical Science 27:1 (2020). Worldwide, at least 570 therapeutic mAbs are being studied in clinical trials, and as of December 2019, 79 therapeutic mAbs had 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. Id.

[0005] Often, therapeutic proteins (e.g., antibodies) are produced in cell cultures obtained from mammalian cells engineered and / or selected to produce high levels of the polypeptide of interest. In fact, mammalian cells are favored over other recombinant protein expression systems for all approved biopharmaceuticals based on recombinant proteins (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 for the expression of recombinant biopharmaceuticals, and the CHO-based system contributes the largest percentage (about 84%) (Tripathi, N.K. et al., Front Bioeng Biotechnol. 7: 420 (2019)).

[0006] Although at a much lower frequency than other cell lines for producing therapeutic antibodies, rat hybridoma cell lines are also used. For example, the rat hybridoma cell line, YB2 / 0, has been used for the production of anti-Rh(D) monoclonal antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function that can be used to prevent 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 des Biotechnologies, is directed to monoclonal antibodies against the CD20 antigen produced by, among other cell lines, the rat hybridoma YB2 / 0 cell line. These patents are hereby incorporated by reference in their entirety. However, there is still a need to produce monoclonal antibodies in rat hybridoma cells on a commercial scale.

[0007] In mammalian cell culture, media formulations, cell lines, 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). The evaluation and understanding of culture process parameters, such as culture pH, dissolved CO2 (pCO2), temperature, and the characterization of the culture medium, in terms of process performance and product quality attributes are the main reasons why these relationships are investigated in process characterization studies and tracked in the manufacturing and quality control processes (good manufacturing process) (GMP) manufacturing of pharmaceuticals and medical devices. Also, the relationships that exist in one cell line do not necessarily apply to another cell line. Jiang, R. et al., Bioprocess Biosyst Eng 41:1731-1741 (2018). Process parameter acceptance limits are defined by manufacturers of therapeutic proteins to ensure that products with desired specifications are consistently achieved.

[0008] 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 in recombinant therapeutic glycoproteins, particularly antibodies. N-linked protein glycosylation typically consists of five major sugars (Figure 1), and it has been shown to play a decisive role in the physiochemical, pharmacokinetic, immunogenic, and Fc effector functions of the protein to which they are attached. Therefore, protein glycosylation (e.g., fucosylation) is usually categorized as a critical quality attribute (CQA) in bioprocessing and is closely monitored in manufacturing to ensure compliance with the acceptance limits defined by the sponsor.

[0009] Optimization of mammalian cell culture process parameters is important for the successful, cost-effective and reproducible commercial production of recombinant proteins with desired specifications, increased titers, high product quality profiles (e.g., low fucosylation) and robust biological activity. Thus, there is a need to improve both recombinant protein productivity and product quality in mammalian cell expression systems, particularly for therapeutic monoclonal antibodies produced at commercial scale. SUMMARY OF THE INVENTION

[0010] Provided herein are methods for generating recombinant proteins (e.g., monoclonal antibodies) in a rat hybridoma cell line, e.g., YB2 / 0, which is a mammalian expression system not commonly used. Also provided are methods for increasing the product titer of such recombinant proteins and improving product quality and functional activity by modifying cell culture process parameters and / or cell culture media. Methods for commercial scale production (e.g., 10,000 L to 25,000 L) of recombinant proteins (e.g., monoclonal antibodies) in rat hybridoma cells that maintain high protein quality and functional activity are also provided. In certain embodiments, the methods of the disclosure are used for the production of anti-CD20 antibodies at commercial scale. Also provided herein are compositions made according to the methods disclosed herein, including pharmaceutical compositions. In some embodiments, the compositions made according to the methods disclosed herein exhibit unique glycosylation patterns that contribute to consistent product quality, clinical safety and efficacy.

[0011] Accordingly, provided herein is a method for producing at least 10,000 L of antibody protein in rat hybridoma cells by culturing the rat hybridoma cells in cell culture having a culture pH of from about 6.5 to about 7.55, wherein the rat hybridoma cells comprise an expression vector comprising a polynucleotide encoding the antibody protein.

[0012] In some embodiments, the culture pH is from about 6.5 to about 7.0. In some embodiments, the culture pH of from about 6.5 to about 7.0 is set on the second day of cell culture. In some embodiments, the culture pH of from about 6.5 to about 7.0 is set on the third day of cell culture.

[0013] In some embodiments, the culture pH is from about 7.0 to about 7.55. In some embodiments, the culture pH of from about 7.0 to about 7.55 is set on days 0 - 3 of cell culture.

[0014] In some embodiments, the culture pH is decreased to from about 6.5 to about 7.0 on the second or third day of cell culture. In some embodiments, the culture pH is decreased on the third day of cell culture. In some embodiments, the culture pH of from about 6.5 to about 7.0 is maintained from the third day of cell culture until collection.

[0015] In some embodiments, the cumulative culture time during which the pH is allowed to drop below the fixed pH set point after the pH has been decreased on the third day, and the cumulative magnitude of the drop (integrated pH2 difference) are smaller compared to cell culture with a larger integrated pH2 difference. In some embodiments, the fixed pH set point is pH 6.91.

[0016] In some embodiments, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and higher titer at collection. In some embodiments, a lower integrated pH2 difference further results in a lower percent fucosylation.

[0017] In some embodiments, rat hybridoma cells expressing an antibody protein are cultured in a chemically defined and animal - derived component - free (ADCF) culture medium.

[0018] In some embodiments, compared to cell culture under the same culture conditions except that the culture pH is 6.60 - 6.8, when the culture pH is 6.6 - 6.96, the harvest titer of the antibody protein increases and / or the fucosylation of the antibody protein decreases.

[0019] In some embodiments, the method further comprises the step of controlling the culture pCO2 level to less than about 300 mmHg. In some embodiments, a pCO2 level of less than about 300 mmHg is facilitated by supplementing the cell culture with additional buffer, increasing the air injection rate, increasing the dissolved oxygen (DO) set point, and / or decreasing the agitation rate.

[0020] In some embodiments, the method further comprises an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture. In some embodiments, the method further comprises a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture.

[0021] In some embodiments, the end of day 1 of the culture is 17 - 33 hours after the start of the cell culture.

[0022] In some embodiments, the method further comprises a third temperature set point of about 32°C to about 33°C, which is set on day 3 of the culture and maintained until harvest. In some embodiments, the third temperature set point is 32.5°C.

[0023] In some embodiments, the cell culture is under the following culture conditions: i) an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture; a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5°C, which is set on day 3 of the culture 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.

[0024] In some embodiments, the yield of the antibody protein is 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% increased compared to the antibody protein produced by a culture process that does not use the culture conditions recited herein.

[0025] In some embodiments, the method further comprises the step of collecting the antibody protein produced by rat hybridoma cells.

[0026] In some embodiments, the method further comprises the step of 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.

[0027] In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (e.g., monoclonal antibody) is an anti-CD20 antibody.

[0028] In some embodiments, the monoclonal antibody is subjected to a CD20, FcγRIIIa-158V and / or C1q binding assay.

[0029] In some embodiments, the monoclonal antibody has a relative potency of 82% to 138% in a cell-based CD20 binding activity bioassay compared to that of a commercial reference standard. In some embodiments, CD20 binding is determined by the binding of an anti-CD20 antibody to the CD20-expressing human mantle cell lymphoma cell line, Jeko-1.

[0030] In some embodiments, the percentage of FcγRIIIa-158V binding is about 82% to about 130% relative to the commercial reference standard for the binding assay. In some embodiments, the percentage of FcγRIIIa-158V binding is determined by surface plasmon resonance (SPR).

[0031] 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 that of 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 that of a commercial reference standard.

[0032] 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 that of a commercial reference standard.

[0033] In some embodiments, the produced monoclonal antibody has a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity compared to monoclonal antibodies produced by a culture process that does not use the culture conditions recited herein. In some embodiments, the monoclonal antibody has a relative potency of 90% to 163% in a cell-based ADCC assay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibody has a relative potency of about 117% in a cell-based ADCC assay compared to that of a commercial reference standard.

[0034] In some embodiments, the monoclonal antibody comprises a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth 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 set forth in SEQ ID NO: 7, and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0035] 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.

[0036] In some embodiments, the cell culture is performed in a bioreactor. In some embodiments, the bioreactor is a commercial-scale bioreactor. In some embodiments, the commercial-scale bioreactor is a 10,000 L, 15,000 L, 20,000 L or 25,000 L bioreactor. In some embodiments, the commercial-scale bioreactor is a 15,000 L bioreactor.

[0037] In some embodiments, the rat hybridoma cell is a YB2 / 0 rat hybridoma cell.

[0038] A method for producing an antibody protein in the culture of rat hybridoma cells on a commercial scale, comprising: a) preparing and thawing a working rat hybridoma cell bank of the target antibody protein; b) culturing the rat hybridoma cells obtained from the cell bank in a series of shaking flasks (125 mL, 500 mL, 3 L, 3 × 3 L shaking flasks and 50 L cell bags) in terms of size and volume, though, to increase by at least 0.30×10 6 viable cells / mL at a target seeding density; c) processing the cell culture with a series of seed bioreactors (120 L, 600 L and 3,000 L) to further increase the volume and cell culture mass; d) inoculating the cell culture from the 3,000 L seed bioreactor into a commercial-scale production bioreactor; e) collecting 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 antibody protein by protein A capture column chromatography; h) inactivating viral factors by solvent-detergent virus inactivation (SDVI). Also provided herein is a method comprising these steps.

[0039] In some embodiments, the commercial-scale production bioreactor is operated in fed-batch mode.

[0040] In some embodiments, a drill hole (10) gas sparger having a 4.0 mm orifice diameter is used in the commercial-scale production bioreactor.

[0041] In some embodiments, the method further comprises purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX).

[0042] In some embodiments, the method further comprises virus filtration (VF) to remove potential viruses.

[0043] In some embodiments, the method further includes ultrafiltration / diafiltration (UFDF).

[0044] In some embodiments, the method further includes preparing a bulk drug substance formulation comprising an antibody protein by adding polysorbate 80 in a formulation buffer to prepare the bulk drug substance formulation. In some embodiments, the method further includes subjecting the bulk drug substance formulation to 0.2 μm filtration. In some embodiments, the method further includes 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. In some embodiments, the antibody protein in the bulk drug substance formulation is formulated into a pharmaceutically acceptable formulation. In some embodiments, the method further includes testing an unprocessed bulk harvest obtained from a commercial-scale production bioreactor for microbial and viral adventitious agents. In some embodiments, the method further includes removing and / or inactivating microbial and viral adventitious agents from a commercial-scale production bioreactor.

[0045] In some embodiments, the rat hybridoma cells are YB2 / 0 cells.

[0046] In some embodiments, the antibody protein is a monoclonal antibody. In some embodiments, the antibody protein (e.g., monoclonal antibody) is an anti-CD20 antibody.

[0047] In some embodiments, the monoclonal antibody comprises a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0048] In some embodiments, the monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0049] In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0050] In some embodiments, the antibody protein comprises an N-glycan profile that includes one or both of the following: i) about 10-20% galactosylated glycan; and / or ii) about 20-40% fucosylated glycan.

[0051] In some embodiments, the N-glycan profile includes about 10-20% galactosylated glycan and about 23%-36% fucosylated glycan. In some embodiments, the N-glycan profile includes about 23%-about 36% fucosylated glycan. In some embodiments, the N-glycan profile includes about 16%-about 18% galactosylated glycan. In some embodiments, the N-glycan profile includes about 17% galactosylated glycan.

[0052] In some embodiments, the antibody protein comprises an N-glycan profile that includes at least about 10% bisecting N-glycan. In some embodiments, the N-glycan profile includes about 12%-about 30% bisecting N-glycan. In some embodiments, the N-glycan profile includes about 18% bisecting N-glycan.

[0053] In some embodiments, the antibody protein comprises an N-glycan profile that contains less than 5% sialylated glycan. In some embodiments, the N-glycan profile contains less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycan. In some embodiments, the N-glycan profile contains no detectable amount of sialylated glycan.

[0054] In some embodiments, the antibody protein comprises an N-glycan profile that contains 0.1% - 1.5% Man5 N-glycan. In some embodiments, the N-glycan profile contains 0.4% - 0.7% Man5 N-glycan. In some embodiments, the N-glycan profile contains approximately 0.6% Man5 N-glycan. In some embodiments, Man5 N-glycan is the only high-mannose species in the N-glycan profile.

[0055] In some embodiments, the antibody protein is produced on a commercial scale of about 10,000 L to about 25,000 L. In some embodiments, the commercial scale is 15,000 L.

[0056] In some embodiments, the method yields an antibody protein harvest titer 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.

[0057] 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., monoclonal antibody) is an anti-CD20 antibody.

[0058] Also provided herein is a rat hybridoma master cell bank (MCB) composition comprising an antibody protein having at least two of the following parameters: i) about 11 - about 13×10 6Peak viable cell density of cells / mL; ii) Harvest titer of about 650 to about 720 mg / L; iii) Percent fucosylation of about 30% to about 38%; iv) About 97% to about 99% monomer detected by size exclusion chromatography (SEC); v) About 1.5% to about 2% dimer detected by SEC; vi) Aggregates at a level of not detectable to about 3% detected by SEC; vii) Fragments at a level of not detectable to about 1% detected by SEC; viii) About 25% to about 30% acidic isoform detected by imaging capillary isoelectric focusing (iCIEF); ix) About 38% to about 49% major isoform detected by iCIEF; and / or x) About 20% to about 36% basic isoform detected by iCIEF.

[0059] A rat hybridoma working cell bank (WCB) composition comprising an antibody protein having at least two of the following parameters is also provided herein: i) Peak viable cell density of about 11 to about 28×10 6 viable cell density of cells / mL; ii) Harvest titer of about 420 to about 1280 mg / L; iii) Percent fucosylation of about 18% to about 40%; iv) About 97% to about 99% monomer detected by size exclusion chromatography (SEC); v) About 1% to about 2% dimer detected by SEC; vi) Aggregates at a level of not detectable to about 2% detected by SEC; vii) Fragments at a level of not detectable to about 1% detected by SEC; viii) About 19% to about 31% acidic isoform detected by imaging capillary isoelectric focusing (iCIEF); ix) About 34% to about 62% major isoform detected by iCIEF; and / or x) About 14% to about 38% basic isoform detected by iCIEF.

[0060] 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., monoclonal antibody) is an anti-CD20 antibody.

[0061] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0062] In some embodiments, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0063] Also provided herein is a method for producing an antibody protein by using the MCB or WCB composition described herein.

[0064] 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.

[0065] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0066] In some embodiments, the anti-CD20 antibody comprises a heavy chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-CD20 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0067] In some embodiments, a method of producing a recombinant protein in rat hybridoma cells comprises culturing rat hybridoma cells in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cells comprise an expression vector comprising a polynucleotide encoding the recombinant protein. In some aspects, the culture pH is from about 6.5 to about 7.0. In some aspects, the culture pH of about 6.5 to about 7.0 is set on the second day of culturing the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is set on the third day of culturing the cell culture. In some aspects, the culture pH is from about 7.0 to about 7.55. In some aspects, the culture pH of about 7.0 to about 7.55 is set on days 0 to 3 of culturing the cell culture.

[0068] In some embodiments, the culture pH is decreased to about 6.5 to about 7.0 on the second or third day of the cell culture. In some embodiments, the culture pH is decreased on the third day of the cell culture. In some embodiments, the culture pH of about 6.5 to about 7.0 is maintained from the third day of culturing the cell culture until collection.

[0069] In some embodiments, the cumulative culture time during which the pH can drop below a fixed pH set point after the pH has been decreased on day 3, and the cumulative magnitude of the drop (integrated pH2 difference) are smaller compared to cell culture with a larger integrated pH2 difference. In some aspects, the fixed pH set point is pH 6.91. 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 percent fucosylation.

[0070] In some embodiments, rat hybridoma cells expressing a recombinant protein are cultured in a culture medium with a known composition and free of animal-derived components (ADCF).

[0071] In some embodiments, compared to cell culture under the same culture conditions except that the culture pH is 6.60 - 6.8, when the culture pH is 6.6 - 6.96, the harvest titer of the recombinant protein increases and / or the fucosylation of the recombinant protein decreases.

[0072] In some embodiments, a method of producing a recombinant protein in rat hybridoma cells further includes the step of controlling the culture pCO2 level to less than about 300 mmHg. In some aspects, a pCO2 level of less than about 300 mmHg is facilitated by supplementing the cell culture with additional buffer, increasing the air injection rate, increasing the dissolved oxygen (DO) set point, and / or decreasing the agitation rate.

[0073] In some embodiments, a method for producing a recombinant protein in rat hybridoma cells further includes an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture. In some aspects, a method for producing a recombinant protein in rat hybridoma cells further includes a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture. In some aspects, the end of day 1 is 17 to 33 hours after the start of the cell culture.

[0074] In some embodiments, a method for producing a recombinant protein in rat hybridoma cells further includes a third temperature set point of about 32°C to about 33°C, which is set on day 3 of the culture and maintained until collection. In some aspects, the third temperature set point is 32.5°C.

[0075] In some embodiments, cell culture in the methods disclosed herein includes the following culture conditions: i). an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture; a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5°C, which is set on day 3 of the culture and maintained until collection; 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 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% increased compared to the recombinant protein produced by a culture process that does not use the culture conditions in i)., ii). and iii).

[0076] In some embodiments, a method of making a recombinant protein in rat hybridoma cells further includes the step of collecting the recombinant protein produced by the rat hybridoma cells. In some aspects, the method further includes the step of purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some aspects, the affinity chromatography includes protein A purification.

[0077] In some embodiments, the purified recombinant protein produced by the rat hybridoma cells is formulated into a pharmaceutically acceptable formulation.

[0078] 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.

[0079] In some embodiments, the monoclonal antibody is subjected to CD20, FcγRIIIa-158V and / or C1q binding assays. In some aspects, the monoclonal antibody has a relative potency of 82% to 138% in a cell-based CD20 binding activity bioassay compared to that of a commercial reference standard. In some aspects, CD20 binding is determined by the binding of an anti-CD20 antibody to the CD20-expressing human mantle cell lymphoma cell line, Jeko-1. In some aspects, the percentage of FcγRIIIa-158V binding of the monoclonal antibody is from about 82% to about 130% relative to a commercial reference standard for the binding assay. In some aspects, the percentage of FcγRIIIa-158V binding is determined by surface plasmon resonance (SPR). In some aspects, 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 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 that of a commercial reference standard. In some aspects, the produced monoclonal antibody has a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity compared to a monoclonal antibody produced by a culture process that does not use culture conditions of i) an initial temperature set point of about 37 °C, which is set on day 0 to day 1 of culture; a second temperature set point of about 35 °C, which is set at the end of day 1 to day 3 of culture; and a third temperature set point of about 32.5 °C, which is set on day 3 of culture and maintained until collection; 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 monoclonal antibody has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard.In some embodiments, the monoclonal antibody has a relative potency of about 117% in a cell-based ADCC assay compared to a commercial reference standard.

[0080] In some embodiments, the monoclonal antibody produced by the methods disclosed herein comprises a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth 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 set forth in SEQ ID NO: 7, and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain having the amino acid sequence set forth in SEQ ID NO: 9. 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.

[0081] In some embodiments, the cell culture is performed in a bioreactor. In some embodiments, the bioreactor is a commercial-scale bioreactor. In some embodiments, the commercial-scale bioreactor is a 10,000 L, 15,000 L, 20,000 L or 25,000 L bioreactor. In some embodiments, the commercial-scale bioreactor is a 15,000 L bioreactor.

[0082] In some embodiments, the rat hybridoma cells expressing the recombinant protein are YB2 / 0 rat hybridoma cells.

[0083] Also provided herein are recombinant proteins made according to any of the methods disclosed herein. In some embodiments, the recombinant protein is a monoclonal antibody.

[0084] A method for producing a recombinant protein in the culture of rat hybridoma cells on a commercial scale, comprising: a) preparing and thawing a working rat hybridoma cell bank of the recombinant protein of interest; b) culturing the rat hybridoma cells obtained from the cell bank to an at least 0.30×10 6 cells / mL target seeding density using a series of shake flasks (125 mL, 500 mL, 3 L, 3×3 L shake flasks and 50 L cell bags) by size and volume; c) processing the cell culture using a series of seed bioreactors (120 L, 600 L and 3,000 L) to further increase the volume and cell culture mass; d) inoculating the cell culture from the 3,000 L seed bioreactor into a commercial scale production bioreactor; e) collecting the cell culture supernatant from the commercial scale production bioreactor; f) clarifying the harvested cells by continuous centrifugation followed by depth filtration; g) purifying the recombinant protein by protein A capture column chromatography; and h) inactivating viral agents by solvent-detergent virus inactivation (SDVI). Also provided herein. In some embodiments, the commercial scale production bioreactor is operated in fed-batch mode. In some embodiments, a drill hole (10) gas sparger having a 4.0 mm orifice diameter is used in the commercial scale production bioreactor.

[0085] In some embodiments, a method for commercial scale production of a 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 a step of preparing a bulk drug substance formulation containing a recombinant protein, including adding polysorbate 80 in a formulation buffer to prepare the bulk drug substance formulation. In some aspects, the method further includes subjecting the bulk drug substance formulation to 0.2 μm filtration. In some aspects, the method further includes 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. In some aspects, the recombinant protein in the bulk drug substance formulation is formulated into a pharmaceutically acceptable formulation. In some aspects, the method further includes a step of testing an unprocessed bulk harvest obtained from a commercial scale production bioreactor for microbial and viral adventitious agents. In some aspects, the method further includes a step of removing and / or inactivating microbial and viral adventitious agents from a commercial scale production bioreactor.

[0086] In some embodiments, in a method for commercial scale production of a recombinant protein, the rat hybridoma cells are YB2 / 0 cells.

[0087] 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 includes 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile that includes one or both of the following: i) about 10-20% galactosylated glycan; and / or ii) about 20-40% fucosylated glycan. In some embodiments, the N-glycan profile includes about 10-20% galactosylated glycan and about 20-40% (e.g., about 23-36%) fucosylated glycan. In some embodiments, the N-glycan profile includes about 23% to about 36% fucosylated glycan. In some embodiments, the N-glycan profile includes about 16% to about 18% galactosylated glycan. In some embodiments, the N-glycan profile includes about 17% galactosylated glycan.

[0092] In some embodiments, the recombinant protein or monoclonal antibody produced by the methods disclosed herein comprises an N-glycan profile that includes at least about 10% bisecting N-glycan. In some embodiments, the N-glycan profile includes about 12% to about 30% bisecting N-glycan. In some embodiments, the N-glycan profile includes about 18% bisecting N-glycan.

[0093] In some embodiments, the recombinant proteins or monoclonal antibodies produced by the methods disclosed herein comprise an N-glycan profile that contains less than 5% sialylated glycans. In some embodiments, the N-glycan profile contains less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some embodiments, the N-glycan profile does not contain a detectable amount of sialylated glycans.

[0094] In some embodiments, the recombinant proteins or monoclonal antibodies produced by the methods disclosed herein comprise an N-glycan profile that contains 0.1% - 1.5% Man5 N-glycans. In some embodiments, the N-glycan profile contains 0.4% - 0.7% Man5 N-glycans. In some aspects, the N-glycan profile contains approximately 0.6% Man5 N-glycans. In some embodiments, the Man5 N-glycans are the only high-mannose species in the N-glycan profile.

[0095] In some embodiments, the recombinant proteins or monoclonal antibodies are produced at a commercial scale of about 10,000 L to about 25,000 L. In some embodiments, the commercial scale is 15,000 L.

[0096] In some embodiments, the disclosed methods yield a recombinant protein or monoclonal antibody harvest titer 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.

[0097] Also provided herein are rat hybridoma master cell bank (MCB) compositions and rat hybridoma working cell bank (WCB) compositions that can be used for the production of the recombinant proteins (e.g., monoclonal antibodies) disclosed herein.

[0098] In some embodiments, the rat hybridoma MCB provided herein comprises a recombinant protein 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) a peak viable cell density of about 11 to about 13×10 6 viable cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a percent fucosylation of about 30% to about 38%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimer as detected by SEC; vi) undetectable to about 3% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 25% to about 30% acidic isoform as detected by imaging 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.

[0099] In some embodiments, the rat hybridoma MCB provided herein comprises a recombinant protein 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) a peak viable cell density of about 11 to about 13×10 6 viable cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a percent fucosylation of about 30% to about 38%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimer as detected by SEC; vi) undetectable to about 3% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 25% to about 30% acidic isoform as detected by imaging 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.

[0100] In some embodiments, the rat hybridoma WCB provided herein comprises a recombinant protein having at least two of the following parameters: i) a peak viable cell density of about 11 to about 28×10 6 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a percent fucosylation of about 18% to about 40%; iv) about 97% to about 99% monomer detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimer detected by SEC; vi) undetectable to about 2% level of aggregates detected by SEC; vii) undetectable to about 1% level of fragments detected by SEC; viii) about 19% to about 31% acidic isoform detected by imaging capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoform detected by iCIEF; and / or x) about 14% to about 38% basic isoform detected by iCIEF.

[0101] In some embodiments, the rat hybridoma WCB provided herein comprises a recombinant protein having at least two of the following parameters: i) a peak viable cell density of about 11 to about 28×10 6 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a percent fucosylation of about 18% to about 40%; iv) about 97% to about 99% monomer detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimer detected by SEC; vi) undetectable to about 2% level of aggregates detected by SEC; vii) undetectable to about 1% level of fragments detected by SEC; viii) about 19% to about 31% acidic isoform detected by imaging capillary isoelectric focusing (iCIEF); ix) about 34% to about 62% major isoform detected by iCIEF; and / or x) about 14% to about 38% basic isoform detected by iCIEF.

[0102] In some embodiments, the rat hybridoma cells in the MCB composition or the 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.

[0103] 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.

[0104] As disclosed herein, provided is also a method of making a recombinant protein by using an MCB or WCB composition. In some embodiments, 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 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.

Brief Description of the Drawings

[0105]

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MODE FOR CARRYING OUT THE INVENTION

[0106] Provided herein is a method for producing a recombinant protein (e.g., a monoclonal antibody) in a rat hybridoma cell, a mammalian expression system not generally used, e.g., YB2 / 0. Also provided are methods for increasing the product titer of a recombinant protein and improving reproducibility, homogeneity, product quality (e.g., low percent fucosylation), and functional activity by modifying cell culture process parameters and / or the 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 a recombinant protein (e.g., a monoclonal antibody) in a rat hybridoma cell that retains high protein quality and functional activity. Also provided herein are compositions made according to the methods disclosed herein, including pharmaceutical compositions.

[0107] In some embodiments, the methods of the present disclosure are used to produce anti-CD20 antibodies at commercial scale (e.g., 10,000L - 25,000L). In some aspects, the anti-CD20 antibodies produced by the manufacturing processes described herein have a unique glycosylation profile that will be further described below. Without being bound by theory, the relative distribution of various N-glycans, or the individual sugar residues present within such N-glycans, can determine the biological and clinical properties of the anti-CD20 antibodies. See U.S. Patent Application No. 63 / 347,852, entitled "Anti-CD20 Antibody Compositions," filed on June 1, 2022, which is co-pending and co-owned and is hereby incorporated by reference in its entirety.

[0108] Abbreviations Table 1 provides a list of abbreviations used in the present disclosure.

[0109]

Table 1

[0110] Definitions To make the present disclosure more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below, unless otherwise clearly presented herein in another manner. Additional definitions are set forth throughout this application.

[0111] As used herein, the term "and / or" shall be construed to mean each specific disclosure of two designated features or components, whether or not the other is present. Thus, when the term "and / or" is used in a phrase such as "A and / or B" herein, it is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in a phrase such as "A, B and / or C", it 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).

[0112] It is understood that wherever aspects using the language "comprising" are described herein, similar aspects are likewise provided in other respects described from the viewpoints of "consisting of" and / or "consisting essentially of".

[0113] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in this disclosure to those of ordinary skill in the art.

[0114] Units, prefixes and symbols are expressed in their accepted form within the International System of Units (SI). Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure that can be obtained by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0115] The use of the alternative (e.g., “or”) is to be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article “a” or “an” is to be understood to refer to “one or more” of any listed or enumerated components.

[0116] The term “about” is used herein to mean approximately, generally, around, or in the region of. When used in conjunction with a numerical range, the term “about” modifies the range by extending the boundaries above and below the numerical value shown. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of, in the absence of other instructions, up or down (higher or lower), 10 percent.

[0117] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range is to be understood to include any integer value within the recited range, and also, where appropriate, fractions thereof (such as one tenth and one hundredth of an integer), etc.

[0118] As used herein, the terms "seeding", "inoculating" or "sowing" refer to the process of providing a cell culture to a bioreactor (e.g., a production bioreactor) or another vessel. In some embodiments, the cells have been previously expanded in another bioreactor or vessel. In some embodiments, the cells are frozen and thawed immediately prior to being provided to the bioreactor or vessel.

[0119] As used herein, the term "cell culture" refers to a suspension of mammalian cells (e.g., rat hybridoma cells) growing in a culture medium according to the present disclosure. As will be apparent from the context, the term "cell culture" or "in a cell culture" can also refer to a closed enclosure, vessel or bioreactor used for the growth of mammalian cells. See also "bioreactor".

[0120] As used herein, the term "bioreactor" refers to a commercial-scale, large-scale, small-scale or micro-scale vessel used to grow mammalian cells (e.g., rat hybridoma cells) according to the methods of the present disclosure. The bioreactor enables various cell culture parameters to be "controlled" in the cell culture process, including but not limited to circulating loop flow, pH, temperature, overpressure and / or medium perfusion rate. Bioreactors include, for example, commercially available bioreactors, stirred tank bioreactors, air-lift bioreactors, bubble column bioreactors, hollow fiber bioreactors, fluidized bed bioreactors, membrane bioreactors, classical fermenters, benchtop bioreactors, 10-15 ml and 250 mL microscale bioreactors (e.g., (ambr) (Sartorius)), as well as cell culture perfusion systems, and disposable or single-use bioreactors.

[0121] The bioreactor can be of any size useful for culturing cells on the desired scale according to the methods of the present disclosure. For example, in some embodiments, the bioreactor used in the methods of the present disclosure can range from about 250 ml to about 25,000 liters. In some embodiments, the bioreactor has a capacity of 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, 330, 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, 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 any intermediate volume.

[0122] In some embodiments, recombinant protein production is carried out on a commercial scale. In certain embodiments, a commercial scale bioreactor can be 10,000L - 25,000L. Thus, as used herein, the term "commercial scale production" of a recombinant protein (e.g., an antibody) refers to production in a bioreactor of at least 10,000L, at least 15,000L, at least 20,000L or at least 25,000L. In some embodiments, the commercial scale bioreactor is 15,000L.

[0123] Suitable bioreactors can be constructed of any material suitable for maintaining cell cultures under the culture conditions of the present disclosure and that aids in cell growth and viability. For example, the bioreactors used in the methods of the present disclosure can be made of glass, plastic, or metal. Suitable bioreactors are known in the art and are commercially available.

[0124] As used herein, the term "production bioreactor" refers to any container between 15 mL and 25,000 L, made of glass, plastic, or metal, that supports a culture environment that aids in cell growth. For example, in Example 3, the production bioreactor is a 15,000 L stainless steel bioreactor. In some embodiments, such as those described in Example 3, the term "production bioreactor" (or N culture vessel) refers to the final bioreactor in a series of increasing scale containers (N-4, N-3, N-2, and N-1 containers or "seed bioreactors", etc.) that are inoculated with a cell culture (also referred to as a "seed culture") from the N-1 container immediately prior to having a high viable cell density, and where cells continue to grow until collection. In some embodiments, cells in the cell culture are collected from the production bioreactor (see, e.g., Example 3).

[0125] As used herein, the term "seed bioreactor" refers to any cell culture container used prior to the transfer of a cell culture to a production bioreactor (see, e.g., Example 3).

[0126] As used herein, the terms "control", "controlled", or "controlling" refer to the ability to intentionally increase, decrease, or maintain protein structure / function parameters. In the methods of the present disclosure, culture pH and temperature are examples of cell culture parameters that can be controlled.

[0127] As used herein, the term "cell" refers to mammalian cells, cultured cells, host cells, recombinant cells, and recombinant host cells. Such cells are generally cell lines obtained from or derived from mammalian tissue that are capable of growing and surviving 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 are capable of expressing and secreting, or can be molecularly engineered to express and secrete, large amounts of recombinant protein (e.g., antibody) in the culture medium.

[0128] Generally, "cells" are cultured over a continuous number of "culture days" (or alternatively, "process days") until they are harvested, as described herein. In some embodiments, 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.

[0129] As used herein, "Day 1 of culture" refers to approximately 12.0 hours to approximately 35.9 hours after inoculation of cells in the seed culture into the production bioreactor. As used herein, "Day 2 of culture" refers to approximately 36.0 hours to approximately 59.9 hours after inoculation of cells in the seed culture into the production bioreactor. As used herein, "Day 3 of culture" refers to approximately 60.0 hours to approximately 83.9 hours after inoculation of cells in the seed culture into the production bioreactor.

[0130] As used herein, the term "culture condition(s)" refers to cell culture conditions at various levels as described herein, such as pH, temperature, pCO2, or shifts in these levels, that result in increased growth, titer, cell density, cell viability, or improved product quality of a recombinant protein (e.g., antibody) expression compared to a cell culture not cultured under specific culture condition(s).

[0131] As used herein, the terms "medium", "cell culture medium", "basal medium" and "culture medium" are used interchangeably, including their grammatical variants, and refer to a physicochemical, 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., monoclonal antibody). Exemplary cell culture media are described below.

[0132] As used herein, the term "set" or "setting" (e.g., "setting" the pH) refers to the input of a specific value(s) of a process parameter (e.g., temperature, pH, etc.) into a bioreactor or other cell culture vessel control system. See also "setpoint".

[0133] As used herein, "setpoint" refers to the setting of conditions in a bioreactor or other cell culture vessel used to grow cells and / or produce a protein product, unless otherwise indicated. In some embodiments, the setpoint is the setting in the production bioreactor. The setpoint can be established at the onset of cell culture and / or reset to different setpoints during cell culture. For example, in some embodiments, the setpoint can be a "pH setpoint". In some embodiments, the setpoint is a "temperature setpoint". In some embodiments, the setpoint can be maintained throughout the cell culture process. In other embodiments, the setpoint can be maintained until a different setpoint is set. In other embodiments, the setpoint can be changed to another setpoint.

[0134] As used herein, "temperature set point" refers to the temperature setting of a bioreactor (e.g., a production bioreactor) or other cell culture processing vessel used to grow cells and / or produce a protein product. The temperature set point can be established at the onset of cell culture in the production bioreactor, in which case it may be referred to as the "initial temperature set point". Subsequent temperature changes during cell culture after the initial temperature set point are referred to as the second temperature set point and, later, the third temperature set point. The last temperature set point before collection may be referred to as the "final temperature set point". In some embodiments, the process can include an initial temperature set point, a second temperature set point, and a third (and final) temperature set point.

[0135] In some embodiments, the "initial temperature set point" is set on day 0 to day 1 of culture. In some embodiments, the "second temperature set point" is set from the end of day 1 to day 3 of culture. In some embodiments, the "third temperature set point" is set on day 3 of culture and maintained until collection (though).

[0136] As used herein, the term "pH1" refers to the culture pH in the production bioreactor after inoculation of cells in the seed culture into the production bioreactor.

[0137] As used herein, the term "pH2" refers to the culture pH in the production bioreactor after a pH shift to a lower pH on day 3 of culture (about 62 - 77 hours, preferably 72 hours) from the culture pH after inoculation ("pH1").

[0138] As used herein, the term "integrated pH2 difference" is a calculated pH parameter that refers to the cumulative magnitude of the drop in pH, along with the cumulative culture time during which the pH can drop below a fixed pH set point after the pH has been decreased on day 3 of culture. The integrated pH2 difference increases when the culture pH drops below the fixed pH set point over any unit time scale. In some embodiments, the integrated pH2 difference of a cell culture compared to a fixed pH set point (e.g., 6.91) is smaller compared to a cell culture having a larger "integrated pH2 difference". In some embodiments, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD), a higher titer at harvest, and a lower percent fucosylation.

[0139] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. This term refers to the primary structure of a molecule. Thus, this 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 derivatized nucleotide bases. The backbone of a polynucleotide can include sugars and phosphate groups (as can typically be found in RNA or DNA), or modified or substituted sugars or phosphate groups. Polynucleotides can be made by recombinant, enzymatic, or synthetic means, for example, by solid-phase chemical synthesis followed by purification. When referring to the sequence of a polynucleotide or nucleic acid, reference can be made to the sequence or order of the nucleobase portions of the nucleotides covalently linked together or their modifications.

[0140] As used herein, the terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to amino acid polymers of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally or artificially modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.) and other modifications known in the art. Since the polypeptides of the present invention are antibody-based, in some embodiments, it is understood that the polypeptides can occur as single chains or associated chains.

[0141] As used herein, "recombinant protein" refers to a polypeptide or protein produced by recombinant DNA techniques. Polypeptides and proteins produced recombinantly are expressed in a host cell that has been engineered (e.g., a rat hybridoma cell, etc.) when disclosed herein. The term "protein" is intended to include glycoproteins.

[0142] 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 aligning the two sequences and introducing gaps to maximize the identity between the sequences. The alignment can be generated using programs known in the art. For the purposes herein, the alignment of nucleotide sequences can be performed using the blastn program set with default parameters, and the alignment of amino acid sequences can be performed using the blastp program set with default parameters (see National Center for Biotechnology Information (NCBI) at world wide web, ncbi.nlm.nih.gov).

[0143] As used herein, the term "expression vector" refers to any nucleic acid construct containing the elements (e.g., promoter, enhancer) necessary for the transcription and translation of an inserted coding sequence of a polypeptide of interest when introduced into a host cell (e.g., rat hybridoma cells). Expression vectors can include plasmids, phagemids, viruses, and their derivatives. The expression vectors of the present disclosure can include polynucleotides encoding recombinant proteins (e.g., monoclonal antibodies).

[0144] As used herein, the term "glycoprotein" refers to a protein modified by the addition of one or more carbohydrate moieties, such as a polysaccharide or oligosaccharide, attached to the protein via an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine or threonine residue ("O-linked") or an asparagine residue ("N-linked").

[0145] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide, such as a polymer or oligomer composed of monosaccharide residues.

[0146] As used herein, the term "upstream process" refers to activities involved in the production and collection of a protein (e.g., an antibody) from cells (e.g., in cell culture of a recombinant protein) in the context of the preparation of a protein, such as an antibody.

[0147] As used herein, the term "downstream process" refers to one or more techniques used after upstream process technology for purifying a protein of interest, such as an antibody, in the context of the preparation of a protein, such as an antibody. For example, downstream process technology includes, 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 purification of protein products using centrifugation.

[0148] As used herein, the term "glycosylation" in relation to a recombinant glycoprotein refers to the addition of a complex oligosaccharide structure to the protein at specific sites within the polypeptide chain. Protein glycosylation and the subsequent processing of the added carbohydrate can affect protein folding and structure, protein stability including protein half-life, and the functional properties of the protein. Protein glycosylation can be divided into two classes, O-linked glycosylation and N-linked glycosylation, based on the context of the sequence where the modification occurs. O-linked polysaccharides are linked to a hydroxyl group, typically the hydroxyl group of either a serine or threonine residue. O-glycans are not added to all serine and threonine residues. O-linked oligosaccharides are typically monoantennary or biantennary, i.e., contain one or at most two branches (antennas) and include 1 - 4 different types of sugar residues added one by one. N-linked polysaccharides are attached to the amide nitrogen of asparagine. Only asparagine that is part of either of two tripeptide sequences, 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 1 - 4 branches, referred to as monoantennary, biantennary, triantennary, tetraantennary. The structures of the sugar residues found in N- and O-linked oligosaccharides are different. Despite such differences, the terminal residues in each branch of both N- and O-linked polysaccharides can be modified by sialic acid residues. Sialic acid is the general name for a family of unique nine-carbon monosaccharides that can be linked to other oligosaccharides. The two major types of sialyl residues found in biopharmaceuticals produced in mammalian expression systems are N-acetyl-neuraminic acid (NANA) and N-glycolyl-neuraminic acid (NGNA).

[0149] As used herein, the term "sialylation" refers to the addition of a sialic acid (S) residue, e.g., N-glycans: G1FS1, G2S1, G2FS1, G2FBS1, G2S2, G2FS2, G2FBS2, to a recombinant glycoprotein.

[0150] As used herein, the term "fucosylation" or "protein fucosylation" refers to the addition of fucose (F) residues to recombinant glycoproteins, such as N-glycans: G0F-GN, G0F, G0FB, G1F, G1FB and G2F.

[0151] As used herein, the term "galactosylation" refers to the addition of galactose (gal) residues to recombinant glycoproteins, such as N-glycans: G1, G2, G1F, G1FB, G2F.

[0152] As used herein, the term "percent fucosylation" or "% fucosylation" refers to the percentage of N-glycans that carry fucose sugars out of all N-glycans. Similarly, the term "percent galactosylation" or "% galactosylation" refers to the percentage of N-glycans that carry galactose sugars out of all N-glycans. Percent fucosylation is calculated, for example, by subjecting a sample or population of anti-CD20 antibody protein to enzymatic deglycosylation such 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 the N-glycans cleaved using enzymatic digestion.

[0153] 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 microscopically using a commercially available cell counting device or a suitable commercially available probe introduced into the bioreactor itself (or a loop through which the medium and suspended cells are passed and then returned to the bioreactor).

[0154] As used herein, the term "integrated viable cell density" or "IVCD" refers to the viable cell density integrated over the duration of a cell culture.

[0155] As used herein, the term "viable cell density" or "VCD" refers to the number of live cells present in a given volume of media under a given set of experimental conditions.

[0156] 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. This term, as used herein, also refers to the percentage (%) of cells that are alive at a given time point in relation to the total number of cells (e.g., live and dead) in the culture at that time point.

[0157] As used herein, the term "shift" refers to the modulation or change of a particular cell culture parameter (e.g., pH shift, temperature shift).

[0158] As used herein, the term "post - shift" (when preceding a particular numerical value or percentage) indicates that a particular 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) is obtained at any time point after a shift in a particular cell culture parameter.

[0159] As used herein, the "initial growth phase" of a cell culture refers to days 0 - 2 of the culture (i.e., days 0, 1, and / or 2) when 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 on later days in the protein production phase (i.e., days 3 of culture - harvest).

[0160] As used herein, the "protein production phase" of cell culture refers to the period from day 3 of culture until collection, during which rat hybridoma cells (e.g., YB2 / 0) produce recombinant protein in a significantly greater amount than in the initial growth phase such that the titer can be measured.

[0161] As used herein, the term "collection" refers to the point in the mammalian cell culture process at which cells containing recombinant protein are separated and removed from the cell culture medium and subjected to additional processing such as centrifugation, filtration, or purification. In some embodiments, cell collection will occur at either day 12 or 13 of the cell culture process, or earlier, when the cell viability drops below 20%. See also "collection titer".

[0162] As used herein, the term "collection material" refers to cells containing recombinant protein recovered from the production bioreactor at the time of "collection".

[0163] 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.

[0164] As used herein, the term "working cell bank" or "WCB" refers to a cell bank generated under GMP conditions from cells expanded from the MCB.

[0165] 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.

[0166] As used herein, the term "unprocessed bulk" refers to the collection material obtained from the production bioreactor prior to clarification.

[0167] As used herein, the term "harvest clarification" refers to a primary purification stage for the removal of cells, particulates, and impurities from the harvested bioreactor material. See, for example, Example 3. After the harvest clarification stage, the clarified harvest can be stored, for example, 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.

[0168] As used herein, the term "single batch" refers to a composition derived from a single production or run from a specified volume of a single bioreactor in the context of a recombinant protein (e.g., anti-CD20 antibody). For example, an anti-CD20 antibody obtained from a single run of a 15,000 L bioreactor may be referred to as a single batch. In some embodiments, 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 embodiments, the anti-CD20 antibody is present in such a single batch at a concentration of 10 - 35 mg / ml; 10 - 30 mg / ml; 10 - 25 mg / ml; 10 - 20 mg / ml; 10 - 15 mg / ml; 15 - 35 mg / ml; 15 - 30 mg / ml; 15 - 25 mg / ml; 15 - 20 mg / ml; 20 - 35 mg / ml; 20 - 30 mg / ml; 20 - 25 mg / ml; 25 - 35 mg / ml; or 25 - 30 mg / ml. In some embodiments, 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 embodiments, the amount of total protein is quantified by spectrophotometry. In some embodiments, the amount of total protein is quantified by spectrophotometric absorbance at 280 nm.

[0169] As used herein, the term "extraneous substance" refers to microorganisms (e.g., bacteria, fungi, viruses, mycoplasma) that are unintentionally introduced during the manufacturing process of a biological medicinal product and that can pose a risk to human health.

[0170] As used herein, the term "titer" refers to the total amount of a recombinant expressed protein (e.g., an antibody) produced by cell culture divided by a given volume of media. The term "titer" refers to concentration and is typically expressed in units of milligrams (mg) of protein per milliliter (mL) or liter (L) of media. In some embodiments, the methods of the disclosure can substantially increase the protein product titer as compared to protein product titers produced by other cell culture methods known in the art or cell culture methods that do not use the culture conditions described herein.

[0171] As used herein, the term "harvest titer" refers to the total amount of a protein (e.g., an antibody) produced by cell culture at the time of harvesting cells from a cell culture, cell culture vessel, or bioreactor. In some embodiments, harvesting of the cells will occur at either the 12th or 13th day of the cell culture process or earlier when the cell viability drops below 20%.

[0172] As used herein, the term "antibody" or "Ab" includes, without limitation, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (VH, which is abbreviated herein) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each L chain comprises a light chain variable region (VL, which is abbreviated herein) 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 regions of high frequency variability called complementarity determining regions (CDRs) interspersed with more conserved 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 carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0173] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those of skill 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.

[0174] The term "antibody" includes, by way of example, 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. Unless expressly stated otherwise, and unless the context indicates otherwise, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of the immunoglobulins described above, including monovalent and bivalent fragments or portions, as well as single-chain antibodies.

[0175] The term "monoclonal antibody" (mAb) refers to a preparation of antibody molecules of single molecular composition, i.e., a preparation of antibody molecules that are essentially identical in their primary sequences and that exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are an example of isolated antibodies. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic or other techniques known to those of skill in the art.

[0176] As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, a collection of two or more non-contiguous regions of a polypeptide(s) (conformational, non-linear, discontinuous or non-contiguous epitope). Epitopes formed from contiguous amino acids are typically, but not necessarily always, retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. An epitope typically comprises 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 contiguous peptides (e.g., derived from L1CAM) are tested for reactivity with a given antibody (e.g., an anti-L1CAM antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as x-ray crystallography, two-dimensional nuclear magnetic resonance and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). In some embodiments, an antibody can bind to two or more epitopes (e.g., TG-1101).

[0177] "Human antibody" (HuMAb) refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Further, when the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0178] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDRs of a non-human antibody have been replaced with the corresponding amino acids derived from human immunoglobulins. In one aspect of the humanized form of an antibody, some, most or all of the amino acids outside the CDRs have been replaced with amino acids derived from human immunoglobulins, while some, most or all of the amino acids within one or more CDRs are not changed. Minor additions, deletions, insertions, substitutions or modifications of amino acids are acceptable as long as they do not inhibit the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains the same antigen specificity as that of the original antibody.

[0179] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species, such as a mouse antibody, and the constant region is derived from another species, such as a human antibody, i.e., an antibody in which the variable region is derived from one species and the constant region is derived from another species.

[0180] As used herein, "acceptable range" or "AR" refers to a range of values for a particular process parameter (e.g., pH), whereby control within the values ensures that process performance and product quality characteristics meet their specifications. Deviation from the "acceptable range" can result in a formal root cause analysis and impact assessment.

[0181] Method for producing a recombinant protein (e.g., monoclonal antibody) in rat hybridoma cells In one aspect, the present disclosure provides a method for producing a recombinant protein (e.g., monoclonal antibody) in rat hybridoma cells, the method comprising culturing the cells in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cells comprise an expression vector comprising a polynucleotide encoding the recombinant protein. In some aspects, the culture pH is from about 6.5 to about 7.0. In some aspects, the culture pH of about 6.5 to about 7.0 is set on the second day of culturing the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is set on the third day of culturing the cell culture. In some aspects, the culture pH is from about 7.0 to about 7.55. In some aspects, the culture pH is set to about 7.0 to about 7.55 on days 0 to 3 of culturing the cell culture.

[0182] In some aspects, the culture pH is decreased to about 6.5 to about 7.0 on the second or third day of the cell culture. In some aspects, the culture pH is decreased on the third day of the cell culture. In some aspects, the culture pH of about 6.5 to about 7.0 is maintained from the third day of culturing the cell culture until collection.

[0183] The "integrated pH2 difference" is a calculated pH parameter that refers to the cumulative magnitude of the drop in pH as the cumulative culture time during which the pH can drop below a fixed pH set point after the pH has been decreased on the third day of culture. The integrated pH2 difference increases when the culture pH drops below the fixed pH set point over any unit time scale. In some aspects, the integrated pH2 difference of the cell culture compared to the fixed pH set point is smaller compared to a cell culture having a larger "integrated pH2 difference". In some aspects, the fixed pH set point is pH 6.91. In some aspects, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and higher titer at the time of collection. In some aspects, a lower integrated pH2 difference further results in a lower percent fucosylation.

[0184] In some embodiments, rat hybridoma cells expressing recombinant proteins are cultured in an animal-free chemically defined production (AFCP) medium. In some embodiments, the basal culture medium is CDM4Mab™ (Cytiva), and the feed medium is BalanCD CHO Feed 4™ (Irvine Scientific).

[0185] In some embodiments, compared to cell culture under the same culture conditions except that the culture pH is 6.60 - 6.8, when the culture pH is 6.6 - 6.96, the harvest titer of the recombinant protein increases and / or the fucosylation of the recombinant protein decreases.

[0186] In some embodiments, the culture pCO2 level in cell culture is controlled to be less than about 300 mmHg. In some embodiments, a pCO2 level of less than about 300 mmHg is facilitated by supplementing the cell culture with additional buffer, increasing the air injection rate, increasing the dissolved oxygen (DO) set point, and / or decreasing the agitation rate.

[0187] In some embodiments, the culture conditions further include an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture. In some embodiments, the culture conditions further include a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture. In some embodiments, "the end of day 1" is 17 - 33 hours after the start of the cell culture. In some embodiments, the culture conditions further include a third temperature set point of about 32°C to about 33°C, which is set on day 3 of the culture and maintained until harvest. In some embodiments, the third temperature set point is 32.5°C.

[0188] In some embodiments, the present disclosure relates to a method for producing a recombinant protein, wherein the cell culture is under the following culture conditions: i). an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture; a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5°C, which is set on day 3 of the culture and maintained until collection; 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 embodiments, "the end of day 1 of the culture" is 17 to 33 hours after the start of the cell culture.

[0189] In some embodiments, the yield of the recombinant protein is 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% higher compared to the recombinant protein produced by a culture process that does not use: i) an initial temperature set point of about 37°C, which is set on day 0 to day 1 of the culture; a second temperature set point of about 35°C, which is set at the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5°C, which is set on day 3 of the culture and maintained until collection; ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0190] In some embodiments, the method disclosed herein further comprises the step of collecting the recombinant protein produced by rat hybridoma cells. In some embodiments, the step of purifying the recombinant protein is performed by affinity chromatography and / or ion exchange chromatography. In some embodiments, the affinity chromatography includes protein A purification.

[0191] In some embodiments, the purified recombinant protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation. In some embodiments, the quality of the purified recombinant protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis.

[0192] In some embodiments, the recombinant protein is a monoclonal antibody. In some embodiments, the monoclonal antibody specifically binds to an epitope of CD20. In some embodiments, the monoclonal antibody is subjected to various assays to assess its potency and biological activity. In some embodiments, the monoclonal antibody is subjected to CD20, FcγRIIIa-158V and / or C1q binding assays. The results of these bioassays are described below.

[0193] Rat hybridoma cells Many mammalian cells or cell types that permit cell culture and polypeptide expression are known in the art and include, for example, the 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 CRL 1651); human fetal-derived kidney line (293 cells 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-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2), and the like.

[0194] In some embodiments, the mammalian cells used to culture and express recombinant proteins (e.g., monoclonal antibodies) according to the methods of the disclosure are rat hybridoma cells. Exemplary rat hybridoma cells include YB2 / 0, IR983F, IR2, and IR162.

[0195] In one aspect, the rat hybridoma cells used in the methods of the present disclosure are YB2 / 0 rat hybridoma cells (ATCC CRL 1662).

[0196] Recombinant proteins (e.g., therapeutic antibodies) 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, recombinant proteins that can be produced from rat hybridoma cells according to the present disclosure include, for example, any pharmaceutically or commercially relevant antibody, enzyme, receptor, hormone, regulator, antigen, or binder.

[0197] Worldwide, at least 570 therapeutic monoclonal antibodies (mAbs) are being studied in clinical trials by commercial companies, and as of December 2019, 79 therapeutic mAbs have been approved by the US FDA and are currently on the market, including 30 mAbs for the treatment of cancer. Also, there is still significant potential for growth. See Lu, R.-M. et al., Journal of Biomedical Science 27:1 (2020). The top 10 best-selling therapeutic antibody drugs in 2018 were Rituxan: anti-CD20 for non-Hodgkin lymphoma; Herceptin: anti-HER2 for breast cancer; Remicade: anti-TNFα for Crohn's disease; Keytruda: anti-PD-1 for melanoma; Opdivo: anti-PD-1 for melanoma and non-small cell lung cancer (NSCLC); Stelara: anti-IL-12 / 23 for psoriasis; Soliris: anti-C5 for paroxysmal nocturnal hemoglobinuria; Avastin: anti-VEGF for colorectal cancer (CRC); Xolair: anti-IgE for asthma; and Humira: anti-TNFα for rheumatoid arthritis (RA). Ibid.

[0198] Given that a number of antibodies are currently in use or in clinical trials as therapeutic agents, the production of antibodies according to the methods of the present disclosure is a preferred embodiment. Any antibody or antigen-binding fragment thereof that can be produced in rat hybridoma cells may be used according to the present disclosure. In some embodiments, the antibody to be produced is a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a polyclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0199] In some embodiments, the monoclonal, polyclonal, chimeric or humanized antibodies described above can contain amino acid residues that do not occur naturally in any antibody of any species in nature. Such foreign residues can be utilized, for example, to confer novel or modified specificities, affinities or effector functions in monoclonal, chimeric or humanized antibodies.

[0200] Anti-CD20 antibody In one embodiment, the monoclonal antibody produced according to the methods disclosed herein binds 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 is capable of binding to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in the targeting of CD20. The degree of binding of the anti-CD20 antibody to an irrelevant non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured, for example, by 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.

[0201] CD20 is a hydrophobic transmembrane phosphorylated protein that is mainly expressed in human and mouse pre-B cells and mature peripheral B cells. In humans, CD20 is also strongly and homogeneously expressed in most mature B cell malignancies, including, for example, most non-Hodgkin B cell lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). The CD20 antigen is not expressed in 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.

[0202] The chimeric anti-CD20 monoclonal antibody rituximab (Rituxan®) has become the standard treatment for many CD20-positive B cell lymphomas and was the first mAb approved for any tumor indication. Demarest, S.J. et al., mAbs 3:338-351 (2011). Biosimilars of rituximab, including rituximab-abbs (TRUXIMA) and rituximab-pvvr (RUXIENCE®), are currently FDA-approved. The RITUXAN HYCELA® (rituximab and hyaluronidase, human) injection for subcutaneous use was FDA-approved in 2017.

[0203] In addition to rituximab, a number of other anti-CD20 antibodies are also known in the art, including, for example, ublituximab (TG-1101), ofatumumab (HuMax; Intracel), ocrelizumab, belimumab, GA101 (obinutuzumab), AME-133v (Applied Molecular Evolution), ocaratuzumab (Mentrik Biotech), PRO131921, tositumomab, ibritumomab tiuxetan, hA20 (Immunomedics, Inc.), BLX-301 (Biolex Therapeutics), Reditux (Dr. Reddy’s Laboratories), and PRO70769 (described in WO2004 / 056312).

[0204] Rituximab is a genetically engineered chimeric mouse / human monoclonal antibody against the CD20 antigen. Rituximab is the antibody called "C2B8" in U.S. Patent No. 5,736,137. The amino acid sequence of rituximab and exemplary methods for its production by recombinant expression in CHO cells are disclosed in U.S. Patent No. 5,736,137, which is hereby incorporated by reference in its entirety. Rituximab was first approved by the FDA in 1997 for the treatment of non-Hodgkin's lymphoma.

[0205] Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody. Studies have indicated that ofatumumab dissociates from CD20 at a slower rate and binds to a membrane-proximal epitope compared to rituximab. Zhang et al., Mabs 1: 326-331 (2009). Epitope mapping has indicated that ofatumumab binds to an epitope located closer to the N-terminus of CD20 and includes the extracellular loop of the antigen compared to the position targeted by rituximab. Id.

[0206] As used herein, "TG-1101" (TG Therapeutics, Inc.) (also known as oublituximab, UBX, UTX, TG-1101, TGTX-1101, Utuxin™, LFB-R603, TG20, EMAB603) is the source antibody of the anti-CD20 antibody described herein having a unique glycosylation profile produced by the methods disclosed herein.

[0207] The supply source antibody, TG-1101, is a monoclonal antibody that targets 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.

[0208] TG-1101 has been studied in various patient cancer populations (e.g., NHL, CLL) both as a single agent and in combination with other agents. For example, O’Connor, O.A. et al., J. Clin. Oncol. 32:5s (2014), (suppl; Abstract No. 8524) showed that TG-1101 was well tolerated and active in rituxin-exposed patients. In a Phase I trial, Lunning, M. et al., American Society of Hematology Annual Meeting and Exposition, December 5-8, 2015, Abstract No. 1538 showed that TG-1101 and TGR-1202 demonstrated activity and a favorable safety profile in relapsed / refractory B-cell NHL and high-risk CLL. Also, 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 TG-1101 in combination with ibrutinib was highly active in patients with relapsed and / or refractory mantle cell lymphoma. Results of several studies involving TG-1101 in combination with other agents (e.g., umbralisib, TG-1701, venetoclax) have been reported in ASH Annual Meeting (December 5-8, 2020), Publications 543, 3137, and 1130.

[0209] In addition, TG-1101 has been studied in Phase 2 and 3 clinical trials for the treatment of relapsing multiple sclerosis (RMS). See, for example, Fox, E. et al., Mult. Scler. 27:420-429 (March 2021), Steinman, L. et al., Neurology 96: (suppl 15) 4494 (2021). See also U.S. Patent Application No. 63 / 303,267, filed January 26, 2022, and U.S. Patent Application No. 63 / 288,350, filed December 10, 2021, which are hereby incorporated by reference in their entirety.

[0210] The amino acid (AA) and nucleotide sequences of the antibody TG-1101 (TG Therapeutics, Inc.) are found in Table 11.

[0211] [Table 2-1]

[0212] [Table 2-2]

[0213] [Table 2-3]

[0214] [Table 2-4]

[0215] In some embodiments, TG-1101 comprises the VH CDR1, CDR2, and CDR3 regions of the sequences of SEQ ID NOs: 1, 2, and 3, and the VL CDR1, CDR2, and CDR3 regions of the sequences of SEQ ID NOs: 4, 5, and 6.

[0216] In some embodiments, TG-1101 comprises a heavy chain (HC) having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7, and a light chain (LC) having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 8.

[0217] In some embodiments, TG-1101 comprises the HC of SEQ ID NO: 7 and the LC of SEQ ID NO: 8.

[0218] In some embodiments, TG-1101 comprises the HC of SEQ ID NO: 7 and the LC of SEQ ID NO: 9.

[0219] In some embodiments, a 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.

[0220] In some embodiments, a monoclonal antibody produced by the methods disclosed herein comprises a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 8.

[0221] In some embodiments, a monoclonal antibody produced by the methods disclosed herein 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.

[0222] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein include a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 8.

[0223] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein include a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0224] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein include a deletion of up to 5 N-terminal residues.

[0225] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein include a deletion of up to 10 N-terminal sequences.

[0226] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein include VH of SEQ ID NO: 10 and VL of SEQ ID NO: 12.

[0227] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein bind to the same epitope as TG-1101 (TG Therapeutics, Inc.). In some embodiments, the monoclonal antibodies produced by the methods disclosed herein are anti-CD20 antibodies (i.e., bind to an epitope of CD20).

[0228] Transfection of expression vectors into rat hybridoma cells A nucleic acid sufficient to achieve expression (typically, an expression vector containing a gene encoding a polypeptide or protein of interest and any operably linked genetic control elements) is introduced into rat hybridoma cells by any of a number of well-known techniques. The term "transfection," as used herein, refers to the introduction of one or more exogenous polynucleotides (e.g., an antibody) into rat hybridoma cells by using physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol. 7: 2031-2034 (1987)).

[0229] For example, as described in Example 3, the expression vector HK463-25 (see Figure 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.

[0230] Screen the cells to determine which of the rat hybridoma cells have actually incorporated the vector and express the polypeptide or protein of interest. Traditional methods for detecting a specific polypeptide or protein of interest expressed by mammalian cells include, but are not limited to, immunohistochemical examination, immunoprecipitation, flow cytometry, immunofluorescence microscopy, SDS-PAGE, Western blot, enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) techniques, bioactivity assays, and affinity chromatography.

[0231] Rat hybridoma cell culture Once cells expressing the polypeptide or protein of interest have been identified, the cells are grown in culture by any of a variety of methods well known to those skilled in the art. Cells expressing the protein of interest are typically grown in a temperature and medium that aids in cell survival, proliferation, and viability. The initial culture volume may be of any size, but is often smaller than the culture volume of the production bioreactor used in the final production of the protein of interest, and frequently, the cells are passaged several times in increasing volumes of bioreactor prior to seeding into the production bioreactor. The cell culture can be stirred or shaken to increase oxygenation of the medium and dispersion of nutrients to the cells. Alternatively or in addition, special infusion devices well known in the art can be used to increase and control oxygenation of the cell culture. In accordance with the present disclosure, those skilled in the art will understand that it may be beneficial to control or regulate certain internal conditions of the bioreactor, including but not limited to, pH, temperature, oxygenation, etc.

[0232] The starting cell density in the production bioreactor may be selected by those skilled in the art. In accordance with the present disclosure, the starting cell density in the production bioreactor can be as low as single cells per culture volume. In some embodiments, the starting cell density in the production bioreactor is about 0.1×10 6 ~ about 10×10 6and can cover the range of individual living cells. In some embodiments, the starting cell density in the production bioreactor can range from about 0.1×10 6 to about 2.0×10 6 cells. In some embodiments, the starting cell density in the production bioreactor is 2×10 2 , 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 , 5×10 6 or 10×10 6 living cells or more per mL.

[0233] The initial and intermediate cell cultures may be grown to any desired density before seeding into the next intermediate or final production bioreactor. It is preferred that the majority of the cells remain alive prior to seeding, although not all or almost all survival is required. In one embodiment of the present disclosure, the cells can be removed from the supernatant, for example, by low-speed centrifugation. It may be desirable to wash the removed cells with a medium to remove any unwanted metabolic waste or medium components before seeding into the next bioreactor. The medium can be the medium in which the cells were previously grown, or a different medium or wash solution selected by the practitioner of the present disclosure.

[0234] Next, the cells can be diluted to a density appropriate for seeding into the production bioreactor. In a preferred embodiment of the present disclosure, the cells are diluted in the same medium used in the production bioreactor. Alternatively, the cells can be diluted in a different medium or solution, for example, if the cells are to be stored for a short period prior to seeding into the production bioreactor, according to the needs and desires of the practitioner of the present disclosure or to accommodate the specific requirements of the cells themselves.

[0235] As described above, once seeded into the production bioreactor, the cell culture enters the "initial growth phase". In certain embodiments, the "initial growth phase" refers to days 0 - 2 of the cell culture (i.e., days 0, 1 and / or 2) when 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 that in the "protein production phase" of the cell culture (i.e., days 3 of culture - harvest). The exact conditions will vary depending on the cell type, the organism from which the cells were obtained, and the nature and characteristics of the recombinant protein expressed.

[0236] Following the "initial growth phase", the "protein production phase" of the cell culture occurs. In certain embodiments, the "protein production phase" refers to days 3 of culture - harvest when the cells (e.g., YB2 / 0) produce recombinant protein in significantly greater amounts than in the initial growth phase such that protein titers can be measured. The exact conditions will vary depending on the cell type, the organism from which the cells were obtained, and the nature and characteristics of the recombinant protein expressed.

[0237] According to the present disclosure, the production bioreactor can be of any volume suitable for the large - scale production of recombinant proteins. In one embodiment, the volume of the production bioreactor is at least 500 liters. In other embodiments, 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. One skilled in the art would be able to recognize and select a bioreactor suitable for use in the practice of the present disclosure. The production bioreactor can be constructed of any material that aids in cell growth and viability and does not interfere with the expression or stability of the polypeptide or protein being produced.

[0238] In some embodiments, the cells are grown in the initial growth phase for a period sufficient to achieve a viable cell density that is a given percentage of the maximum viable cell density that the cells would ultimately reach if allowed to grow unhindered. For example, the cells can be grown for a period sufficient to achieve 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 percent of the maximum viable cell density.

[0239] In some embodiments, the cells are grown for a defined period. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells can be grown in culture for 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 days or more. In some cases, the cells can be grown for one month or more. If the growth of the cells in the seed bioreactor at the initial growth phase temperature is sufficient such that the viable cell density in the production bioreactor at the time of inoculation is already the desired percentage of the maximum viable cell density, the cells will be grown in the production bioreactor for 0 days.

[0240] To increase the oxygen addition and nutrient dispersion to the cells, the cell culture can be agitated or shaken during the initial growth phase. In accordance with the present disclosure, certain internal conditions of the bioreactor, including but not limited to pH, temperature, oxygen addition, etc., can be controlled or adjusted during the initial growth phase. For example, the pH can be controlled by supplying an appropriate amount of acid or base, and the oxygen addition can be controlled by an injection device well known in the art.

[0241] A variety of methods for culturing mammalian cells for the production of recombinant proteins are known in the art by batch, fed-batch, continuous, semi-continuous and perfusion culture modes. See, for example, Willard, S.S., M., Bioprocess Int. 15(3) 38-46 (2017), which discusses culture methods in monoclonal antibody production.

[0242] In a preferred embodiment, rat hybridoma cells are cultured using the fed-batch manipulation mode as described herein. 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 point after the beginning of the culture process. In some embodiments of the fed-batch method, nutrients are added after depletion. Fed-batch cultures can be initiated using a basal culture medium (e.g., CDM4Mab®). The culture medium to which additional components are provided to the culture at some point after the beginning of the culture process is the feed medium (e.g., BalanCD CHO Feed 4®). Fed-batch cultures are typically stopped at some point (based on either the number of days in culture or the earlier of cell viability) and the cells and / or components in the medium are collected and purified.

[0243] Alternative culture modes, such as batch and perfusion manipulation modes, are also contemplated for use in the methods of the present disclosure, although those of skill in the art will understand that some modification may be required using routine experimentation methods. For example, one of skill in the art will understand that in the batch mode, no feed medium is used. As used herein, "batch culture" or "batch manipulation mode" refers to a cell culture mode in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions up to a certain density and then collected and processed as a batch before the nutrients are depleted. As used herein, "perfusion culture" or "perfusion manipulation mode" refers to a cell culture mode having a continuous flow of physiological nutrient solution at a constant rate through or over a population of cells.

[0244] Monitoring of cell culture conditions In some aspects of the methods disclosed herein, certain conditions of cell culture growth are monitored periodically. Monitoring of cell culture conditions enables the practitioner to determine whether the cell culture is producing recombinant protein at sub-optimal levels or whether the culture is about to enter a sub-optimal production phase. To monitor certain cell culture conditions, it may be necessary to remove a small aliquot of the culture for analysis.

[0245] By way of non-limiting example, it may be beneficial or necessary to monitor the temperature, pH, cell density, viable cell density, cell viability, integrated viable cell density, lactate level, ammonium level, weight osmolarity, amount of dissolved oxygen, pCO2 level, glutamine level, glutamate level or glucose level of the cell culture, or the titer of the expressed polypeptide or protein. In some aspects, such parameters are measured on a regular basis. In some aspects, such parameters are measured once or multiple times (i.e., 1, 2, 3, 4, 5 times) per day. In some aspects, such parameters are measured on a daily basis. In some aspects, such parameters are measured every other day. In some aspects, such measurements are obtained during the protein production phase of the cell culture. In some aspects, such measurements are obtained during the early growth phase of the cell culture. In some embodiments, the expression or activity level of the expressed recombinant protein is measured on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 after the start of the cell culture.

[0246] A number of techniques well known in the art would enable one of ordinary skill in the art to measure these conditions. For example, cell density can be measured using a hemocytometer, Coulter counter, or cell density test (CEDEX). Viable cell density can be determined by staining a culture sample with trypan blue. Since only dead cells take up trypan blue, viable cell density can be determined by counting the total number of cells, dividing the number of cells that have taken up 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 levels of lactate, ammonium, or expressed polypeptides or proteins. Alternatively, the levels of expressed polypeptides or proteins can be determined by standard molecular biology techniques such as Coomassie staining of SDS-PAGE gels, Western blotting, Bradford assay, Lowry assay, biuret assay, and UV absorbance. It may also be beneficial or necessary to monitor post-translational modifications of expressed polypeptides or proteins, including phosphorylation and glycosylation.

[0247] Shift in process parameters in rat hybridoma (e.g., YB2 / 0) cell culture 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) being expressed. For example, in some aspects, the cell culture can be shifted by shifting one or more of a number of culture conditions, including, for example, temperature, pH, weight osmolarity, and sodium butyrate levels.

[0248] In some aspects, the following process parameters can be shifted during the protein production phase (day 3 of culture → harvest) of the cell culture. In certain aspects, the process parameters can be shifted during the initial growth phase (day 0 → day 2 of culture) of the cell culture.

[0249] In some embodiments, the process parameter shifts described below do not occur simultaneously. In some embodiments, the process parameter shifts described below can occur simultaneously or 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 apart from each other. In some embodiments, one particular process parameter shift (e.g., temperature) does not routinely precede or proceed to another process parameter shift (e.g., pH). Process parameter shifts, when disclosed herein, can occur alone or in combination.

[0250] Control of pH and pCO2 levels Both pCO2 and the 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 mainly important for the control of pCO2 levels. pH is an important process control parameter. The culture pH is mainly important for 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 the cell culture medium, various buffers are supplemented into the cell culture medium. Sodium bicarbonate is one of the most common ones. Alternative buffer systems containing HEPES are also typically used. See Itagaki, A. et al., Experimental Cell Research 83:351-361 (1974). For sodium bicarbonate buffers, the control of culture pH is typically two-sided. When the pH exceeds the desired value, carbon dioxide is added into the bioreactor to decrease the pH to an acceptable value. When the pH drops below the desired value, a dilute basic solution is typically added into the bioreactor to increase the pH to an acceptable value. Alternative strategies for pH control in mammalian cell bioreactors have been investigated. The choice of pH control methodology and buffer system ultimately depends on the manufacturer of the therapeutic protein. The addition and accumulation of excessive base and CO2 in the bioreactor have been shown to be harmful to the growth of mammalian cells in culture. See deZengotita, V. et al., Cytotechnology 28:213-227 (1998). Therefore, the control of the level of dissolved CO2 (pCO2) and base addition is important for the 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 collection and purification from the cell culture.

[0251] pH In some embodiments, a method of producing a recombinant protein in rat hybridoma cells includes culturing the rat hybridoma cells in a cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cells comprise an expression vector comprising a polynucleotide encoding the recombinant protein. 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 the second day of culturing the cell culture. In some embodiments, the culture pH of about 6.5 to about 7.0 is set on the third day of culturing the cell culture. 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 days 0 to 3 of culturing the cell culture.

[0252] In some embodiments, the culture pH is decreased (or shifted) to about 6.5 to about 7.0 on the second or third day of the cell culture. In some embodiments, the culture pH is decreased on the third day of the cell culture. In some embodiments, the culture pH of about 6.5 to about 7.0 is maintained from the third day of culturing the cell culture until collection. In some embodiments, the timing of the pH shift on the third day occurs in the production bioreactor 62 - 77 hours after inoculation.

[0253] In some embodiments, the harvest titer of the recombinant protein increases and / or the fucosylation of the recombinant protein decreases when the culture pH is 6.6 - 6.96, compared to cell culture under the same culture conditions except that the culture pH is 6.60 - 6.8.

[0254] Another pH-related process parameter being monitored in the rat hybridoma cell culture disclosed herein is referred to as the "integrated pH2 difference". See Example 5. The integrated pH2 difference refers to the cumulative culture time during which the pH can drop below a fixed pH setpoint after the pH is decreased on the third day of the culture, and the cumulative magnitude of such a drop. When the culture pH drops below the fixed pH setpoint over any unit time scale, the integrated pH2 difference increases. The fixed pH setpoint is an assigned value. In some embodiments, the fixed pH setpoint is 6.91. In some embodiments, the integrated pH2 difference of the cell culture compared to the fixed pH setpoint (e.g., 6.91) is smaller compared to the cell culture with a larger integrated pH2 difference. In some embodiments, a lower integrated pH2 difference results in a higher integrated viable cell density (IVCD) and a higher titer at the time of collection. In some embodiments, a lower integrated pH2 difference further results in a lower percent fucosylation.

[0255] pCO2 In a cell culture medium having a buffer based on sodium bicarbonate, CO2 is introduced into the cell culture to control the pH. When the pH increases above the targeted value / range, CO2 is added to reduce the pH. The exogenously added CO2 dissolves in the culture medium, increasing the pCO2 level. In some embodiments, as described herein, based on the control of the culture pH, the culture conditions further include the step of controlling the culture pCO2 level to less than about 300 mmHg. As used herein, "less than about 300 mmHg" means that the pCO2 level can range from 0 to 300 mmHg.

[0256] In order to support higher pH control levels while simultaneously controlling low pCO2 levels, there are multiple cell culture process means other than a higher pH set point. Alternative means for facilitating low pCO2 levels include increasing the buffering capacity of the cell culture medium by supplementing with additional buffers such as HEPES. By reducing the need for CO2 addition to the bioreactor to control pH, this will attenuate the culture-related increase in pCO2 during culturing. Another alternative means for facilitating low pCO2 levels in the bioreactor is to increase the pCO2 stripping capacity of the bioreactor by increasing the air overlay and air injection rate so that high levels can be prevented from being achieved. Increasing the dissolved oxygen (DO) set point and / or decreasing the bioreactor agitation rate will have a similar effect as forcing higher air and O2 flow rates into the bioreactor to maintain DO and will effectively flow excess pCO2 out of the bioreactor.

[0257] In some aspects of the methods disclosed herein, pCO2 levels of less than about 300 mmHg are facilitated by supplementing the cell culture with additional buffer, increasing the air injection rate, increasing the DO set point, and / or decreasing the agitation rate.

[0258] In some aspects, the cell culture is performed in a commercial-scale bioreactor. In some aspects, the commercial-scale bioreactor is a 10,000 L, 15,000 L, 20,000 L or 25,000 L bioreactor. In some aspects, the commercial-scale bioreactor is a 15,000 L bioreactor.

[0259] In some aspects, the rat hybridoma cells are YB2 / 0 rat hybridoma cells.

[0260] In some aspects, the recombinant protein is an IgG1 glycoprotein.

[0261] In some embodiments, the recombinant protein is a monoclonal antibody. In some embodiments, the monoclonal antibody is a chimeric, humanized or human antibody.

[0262] In some embodiments, the monoclonal antibody specifically binds to an epitope of CD20 (i.e., is an anti-CD20 antibody).

[0263] In some embodiments, the monoclonal antibody is TG-1101. In some embodiments, the monoclonal antibody binds to the same epitope as TG-1101.

[0264] In some embodiments, for example, additional cell culture process parameters such as temperature control described below are shifted in conjunction with a shift in culture pH or a surrogate shift in pCO2 to optimally express the recombinant protein (e.g., monoclonal antibody) of interest in rat hybridoma cells.

[0265] Temperature shift In the methods disclosed herein, a temperature shift in cell culture is another culture condition for increasing the expression of recombinant protein, cell density or viability, and product quality in a rat hybridoma cell line. In some embodiments, it may be desirable to use multiple distinct temperature shifts at different time points in cell culture.

[0266] In some embodiments, the temperature shift in cell culture in rat hybridoma cells is carried out in combination with the pH shift described herein. In certain embodiments, the temperature shift in cell culture is carried out in combination with the pH shift and also in combination with the use of a cell culture medium of known composition and ADCF. In some embodiments, the cell culture medium of known composition and ADCF is CDM4Mab®. In some embodiments, the feed medium BalanCD CHO Feed 4® is used in combination with CDM4Mab®.

[0267] In some embodiments, the culture conditions include an initial temperature set point of about 37°C, which is set from day 0 to day 1 of the culture.

[0268] In some embodiments, the culture conditions further include a second temperature set point of about 35°C, which is set from the end of day 1 to day 3 of the culture. As used herein, "the end of day 1" refers to 17 - 33 hours after the start of cell culture.

[0269] In some embodiments, the culture conditions further include a third temperature set point of about 32°C to about 33°C, which is set on day 3 of the culture and maintained until collection. In some embodiments, the third temperature set point is 32.5°C.

[0270] In some embodiments, the cell culture is under the following culture conditions: i). an initial temperature set point of about 37°C, which is set from day 0 to day 1 of the culture; a second temperature set point of about 35°C, which is set from the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5°C, which is set on day 3 of the culture and maintained until collection; ii). a culture pH between about 6.5 and about 7.55; and iii). a culture pCO2 of less than about 300 mmHg.

[0271] In some embodiments, "day 1 of the culture" refers to about 12.0 to about 35.9 hours after inoculation of the cells in the seed culture into the production bioreactor. In some embodiments, "day 2 of the culture" refers to about 36.0 to about 59.9 hours after inoculation of the cells in the seed culture into the production bioreactor. In some embodiments, "day 3 of the culture" refers to about 60.0 to about 83.9 hours after inoculation of the cells in the seed culture into the production bioreactor.

[0272] In some embodiments, the timing of the first temperature shift occurs in the production bioreactor 17 - 33 hours after inoculation. In some embodiments, the timing of the second temperature shift occurs in the production bioreactor 62 - 77 hours after inoculation.

[0273] In some embodiments, the recombinant protein harvest titer in rat hybridoma cells is about 0.5 g / L to about 1.5 g / L when the culture pH is controlled and the temperature is shifted, as described herein. In some embodiments, the recombinant protein harvest titer in rat hybridoma cells is about 0.5 g / L to about 1.3 g / L. In some embodiments, the recombinant protein harvest titer in rat hybridoma cells is about 1.0 g / L to about 1.5 g / L.

[0274] In some embodiments, the yield of the recombinant protein is 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% higher compared to the recombinant protein produced by a culture process that uses i) an initial temperature set point of about 37 °C, which is set on day 0 to day 1 of the culture; a second temperature set point of about 35 °C, which is set at the end of day 1 to day 3 of the culture; and a third temperature set point of about 32.5 °C, which is set on day 3 of the culture and maintained until harvest (though); ii) a culture pH between about 6.5 and about 7.55; and iii) a culture pCO2 of less than about 300 mmHg.

[0275] In some embodiments, the percent fucosylation of the prepared recombinant protein is between about 20% and about 35% when the culture pH is controlled and the temperature is shifted, as described herein. In some embodiments, the percent fucosylation of the prepared recombinant protein is about 20% to about 30% when the culture pH is controlled and the temperature is shifted, as described herein.

[0276] In some embodiments, the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein contains fucosylated glycans between 20% and 40%; fucosylated glycans between 23% and 36%; fucosylated glycans between 28% and 33%; or about 33% or about 36% fucosylated glycans (where “about” means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). The fucosylated glycans are the N-glycans shown in FIG. 1 that carry a fucose residue (shown as an unfilled triangle in FIG. 1). Briefly, a sample or population of anti-CD20 antibody is subjected to enzymatic deglycosylation such 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 the N-glycans cleaved using enzymatic digestion.

[0277] In certain embodiments, the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein contains fucosylated glycans between 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%. In some embodiments, the recombinant protein or anti-CD20 antibody i contains about 36% fucosylated glycans.

[0278] In some embodiments, the sialylation of the recombinant protein or anti-CD20 antibody produced by the methods disclosed herein is about 1% to about 4% when the culture pH is controlled and the temperature is shifted as described herein.

[0279] In some embodiments, the recombinant proteins or anti-CD20 antibodies produced by the methods disclosed herein contain less than 10%, 8%, 5%, 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycan. In some embodiments, the recombinant proteins or anti-CD20 antibodies produced by the methods disclosed herein contain sialylated glycan between 10% and 0.5%; sialylated glycan between 10% and 5%; sialylated glycan between 5% and 0.5%; sialylated glycan between 4% and 0.5%; sialylated glycan between 2% and 0.5%; or undetectable amounts of sialylated glycan. Briefly, samples or populations of anti-CD20 antibodies are subjected to enzymatic deglycosylation such 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 sialylated N-glycans is the percentage of sialylated N-glycans among the N-glycans cleaved using enzymatic digestion.

[0280] In certain embodiments, the recombinant proteins or anti-CD20 antibodies produced by the methods disclosed herein contain sialylated glycan between 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%. In some embodiments, the recombinant proteins or anti-CD20 antibodies do not contain detectable amounts of sialylated glycan.

[0281] In some embodiments, the temperature shift and pH control culture conditions described herein further include culturing cells in a cell culture basal medium of known composition and free of animal-derived components (ADCF), such as CDM4Mab® (Cytiva Lifesciences / GE) and Feed Medium BalanCD CHO Feed 4® (Irvine Scientific). In some embodiments, the feed medium is added to the cell culture about every 48 hours. In some embodiments, a glucose, glutamine, and / or cholesterol lipid solution is added for replenishment of the feed medium in cell culture.

[0282] In some embodiments, a method of producing a recombinant protein in rat hybridoma cells that utilizes cell culture pH control and temperature control and cultures the cells in a cell culture medium of known composition and free of animal-derived components (ADCF), such as CDM4Mab® and Feed Medium BalanCD CHO Feed 4®, as described herein, results in a recombinant protein harvest titer of about 0.5 g / L to about 1.3 g / L. In some embodiments, the cell culture is performed in a bioreactor. In some embodiments, the cell culture is performed in a commercial-scale bioreactor (e.g., 10,000 L to 25,000 L). In some embodiments, the cell culture is performed in a 15,000 L bioreactor.

[0283] Cell Culture Medium and Feed Medium Hundreds of basal cell culture medium formulations for use in mammalian cell culture, including serum-free, peptone-free, animal-derived component-free (ADCF), and / or known composition culture media, are well known in the art and are commercially available. To these basal culture medium formulations, one of ordinary skill in the art will add components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and others, depending on the requirements of the particular type of host cell to be cultured.

[0284] Cell culture media typically contain at least one or more components selected from the following: an energy source (e.g., in the form of a carbohydrate such as glucose); essential amino acids including 20 basic amino acids plus cysteine; vitamins and / or other organic compounds typically required at low concentrations; lipids or free fatty acids (e.g., linoleic acid); and trace elements (e.g., inorganic compounds or naturally occurring elements typically required at very low concentrations, usually in the micromolar concentration range). The medium can be solid, gel-like, liquid, gaseous, or a mixture of phases and materials.

[0285] Some examples of commercially available cell culture basal media are listed in Example 1, Table 2, and include, for example, Hycell medium, IS CHO-SD G10.6 medium, CD Hybridoma, Hybridoma without PFHM-II protein, Ex-Cell CD Hybridoma, UltraDOMA-PF Hybridoma, ProDOMA 1, ProDOMA 1, Liquid Medium (LM)|CDM4Mab and ActiCHO-P.

[0286] Furthermore, the cell culture medium may be supplemented as needed to contain one or more additional components at appropriate concentrations or amounts, as known to and practiced by those skilled in the art. Supplements to support the growth and maintenance of a particular cell culture can be readily determined by those skilled in the art, for example, as described in Barnes et al., Cell 22:649 (1980); in Mammalian Cell Culture, Mather, J. P., ed., Plenum Press, NY (1984); and U.S. Patent No. 5,721,121, etc.

[0287] Exemplary supplements include, but are not limited to, chemical genetic selection agents, hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, somatotropin, pituitary extract, aprotinin); salts (e.g., calcium, magnesium and phosphates) 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); cytoprotective agents (e.g., PLURONIC.RTM. F68) and extracellular matrix proteins (e.g., fibronectin).

[0288] In some embodiments, the culture medium suitable for the methods disclosed herein is supplemented with a feed medium. In some embodiments, the feed medium is a feed medium of known composition. In some embodiments, a feed medium of known composition (or CDFM) or medium (media) refers to a medium containing one or more nutrients whose chemical composition and relative concentrations are known and that is added to the culture medium at some point after inoculation. CDFM is supplied to the culture vessel either continuously or in discrete increments, during culture to the culture medium, with or without periodic cell and / or product harvest before the end of the cell culture. CDFM can be formulated individually to contain a specific blend of amino acids, vitamins, trace minerals and organic compounds in a concentrated amount to function as a feed medium to the cell culture medium. Alternatively, commercially available CDFM may be used. Some examples of commercially available CDFM are listed in Example 1, Table 3 and include EX-CELL Advanced CHO Feed 1 (with glucose); Cell Boost 6; CHO CD Efficient Feed A; BalanCD CHO feed 4; Cell Boost 7A and b; and Cell Boost 3.

[0289] In one aspect, the basal culture medium CDM4Mab® (Cytiva Lifesciences / GE) is used in combination with the feed medium BalanCD CHO Feed 4® (Irvine Scientific) in the culture of rat hybridoma cells.

[0290] As disclosed in Example 1, numerous cell culture medium formulations and feed media were studied to maximize cell growth, cell viability, productivity, percent fucosylation, and Fc effector function of recombinant antibodies derived from rat hybridoma cell lines (e.g., YB2 / 0 cells) in culture.

[0291] In certain aspects, the present disclosure is directed to a method 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.

[0292] In some aspects, the rat hybridoma cells are cultured in a culture medium of known composition that is animal-derived component-free (ADCF).

[0293] In some embodiments, rat hybridoma cells are cultured in CDM4Mab® (Cytiva) and Feed Medium BalanCD CHO Feed 4® (Irvine Scientific). In some embodiments, the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® are used in combination with other commercially available cell culture media or with cell culture media formulated individually for use by rat hybridoma cells. In some embodiments, other basal media (other than CDM4Mab®) are used in the rat hybridoma cell culture described herein. In some embodiments, other feed media (other than or in addition to BalanCD CHO Feed 4®) are used in the rat hybridoma cell culture described herein.

[0294] In some embodiments, a basal medium, such as CDM4Mab®, is present in the cell culture starting on day 0 of the cell culture. In certain embodiments, BalanCD CHO Feed 4® is added on day 3 of the cell culture.

[0295] In some embodiments, a feed medium, such as the BalanCD CHO Feed 4® feed medium, is added to the cell culture every about 48 - 72 hours. In some embodiments, a feed medium, such as the BalanCD CHO Feed 4® feed medium, is added to the cell culture every about 48 hours. In some embodiments, the BalanCD CHO Feed 4® feed medium is added to the cell culture every about 72 hours. In some embodiments, a glucose, glutamine, and / or cholesterol lipid solution is added for the replenishment of the feed medium BalanCD CHO Feed 4® in the cell culture.

[0296] In some embodiments, the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is produced at an increased harvest titer compared to cell cultures not cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4®. In some embodiments, the harvest titer of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is from about 0.3 g / L to about 1.5 g / L. In some embodiments, the harvest titer of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is from about 0.3 g / L to about 1.3 g / L. In some embodiments, the harvest titer of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is from about 0.5 g / L to about 1.0 g / L. In some embodiments, the harvest titer of the recombinant protein is about 1.0 g / L.

[0297] In some embodiments, the percent fucosylation of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is reduced compared to cell cultures not cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4®. In some embodiments, the percent fucosylation of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is reduced to about 18% to about 73%. In some embodiments, the percent fucosylation of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is reduced to about 18% to about 40%. In some embodiments, the percent fucosylation of the recombinant protein cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is reduced to about 18% to about 30%. In some embodiments, the percent fucosylation of the recombinant protein is reduced to about 18%.

[0298] In certain embodiments, the rat hybridoma cell line cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is the YB2 / 0 rat hybridoma cell line.

[0299] In some embodiments, the recombinant protein produced in the rat hybridoma cell line cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is an IgG1 glycoprotein. In some embodiments, the recombinant protein produced in the rat hybridoma cell line cultured in the basal medium CDM4Mab® and the feed medium BalanCD CHO Feed 4® is a monoclonal antibody. In some embodiments, the monoclonal antibody is a chimeric, humanized or human antibody.

[0300] In some embodiments, monoclonal antibodies produced in a rat hybridoma cell line cultured in a basal medium CDM4Mab® and a feed medium BalanCD CHO Feed 4® specifically bind to an epitope of CD20. In some embodiments, monoclonal antibodies produced in a rat hybridoma cell line cultured in a basal medium CDM4Mab® and a feed medium BalanCD CHO Feed 4® are the anti-CD20 antibody TG-1101.

[0301] In some embodiments, the percentage of FcγRIIIa-158V binding to monoclonal antibodies produced in a rat hybridoma cell line cultured in a basal medium CDM4Mab® and a feed medium BalanCD CHO Feed 4® is increased compared to the percentage of FcγRIIIa binding to monoclonal antibodies not cultured in a basal medium CDM4Mab® and a feed medium BalanCD CHO Feed 4®. In some embodiments, the percent FcγRIIIa-158V binding increases by about 5% to about 30%. In some embodiments, the percent FcγRIIIa-158V binding increases by about 20% to about 30%. In some embodiments, the percent FcγRIIIa-158V binding increases by about 20%. In certain embodiments, the percent FcγRIIIa-158V binding is evaluated by surface plasmon resonance (SPR).

[0302] In some embodiments, the percentage of antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody produced in a rat hybridoma cell line cultured in a basal medium CDM4Mab™ and a feed medium BalanCD CHO Feed 4™ is increased compared to the percentage of ADCC activity of a monoclonal antibody not cultured in a basal medium CDM4Mab™ and a feed medium BalanCD CHO Feed 4™. In some embodiments, the percent ADCC activity increases by about 10% to about 70%. In some embodiments, the percent ADCC activity increases by about 50% to about 70%. In some embodiments, the percent ADCC activity increases by about 60% to about 70%. In certain embodiments, the percent ADCC activity is evaluated by a cell-based bioassay.

[0303] In some embodiments, the cell culture performed in a basal medium CDM4Mab™ and a feed medium BalanCD CHO Feed 4™ is carried out in a bioreactor. In some embodiments, the bioreactor is a commercial-scale bioreactor (e.g., 15,000 L, 20,000 L or 25,000 L capacity).

[0304] In some embodiments, as described herein, other cell culture process parameters, such as pH and temperature control, are shifted in conjunction with the use of a basal medium CDM4Mab™ and a feed medium BalanCD CHO Feed 4™ to optimally express a recombinant protein (e.g., an antibody) in a rat hybridoma cell line (e.g., YB2 / 0).

[0305] Collection of recombinant protein, and assay of protein quality, titer and potency In some embodiments, a method of making a recombinant protein further includes collecting the recombinant protein produced by the rat hybridoma cells. In some embodiments, collection of the cell culture will occur at either the 12th or 13th day of cell culture, or earlier if the cell viability drops below 20%.

[0306] Generally, it will typically be desirable to isolate and / or purify the proteins or antibodies expressed in accordance with the present disclosure. In preferred embodiments, the expressed polypeptide or protein is secreted into the medium, and thus, for example, as a first step in the purification process, cells and other solids can be removed by centrifugation, filtration, or the like.

[0307] Alternatively, the expressed polypeptide or protein is bound to the surface of the host cell. In this embodiment, the medium is removed and, as a first step in the purification process, the host cells expressing the polypeptide or protein are lysed. Lysis of mammalian host cells can be accomplished by any of a number of means well known to those of skill in the art, including physical disruption with glass beads, exposure to high pH conditions, exposure to freezing temperatures, and addition of a cell lysis buffer containing a surfactant.

[0308] Polypeptides or proteins can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, or by any other available technique for protein purification (e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N. (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, the protein can be isolated by binding it to an affinity column containing an antibody raised against the protein and attached to a stationary support. Alternatively, an affinity tag such as an influenza coat sequence, polyhistidine, or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over an appropriate affinity column. To reduce or eliminate degradation of the polypeptide or protein during the purification process, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, or aprotinin can be added at any or all stages. Protease inhibitors are particularly desirable if cells need to be lysed to isolate and purify the expressed polypeptide or protein.One of ordinary skill in the art will recognize that the exact purification technique will vary depending on the characteristics of the polypeptide or protein to be purified, the characteristics of the cells in which the polypeptide or protein is expressed, and the composition of the medium in which the cells were grown.

[0309] In some embodiments, the methods disclosed herein further comprise purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some embodiments, the affinity chromatography comprises protein A purification. In some embodiments, the purified recombinant protein produced by rat hybridoma cells is formulated into a pharmaceutically acceptable formulation.

[0310] In some embodiments, the quality of the product is evaluated after the expressed recombinant protein has been collected and purified from the cell culture.

[0311] In some embodiments, the quality of the purified recombinant protein is measured by SEC-HPLC, imaging capillary electrophoresis (ICIEF) and / or N-linked glycan analysis. In some embodiments, the purified recombinant protein (e.g., monoclonal antibody) is measured for percent fucosylation by performing N-linked glycan analysis as described, for example, in Examples 1, 2, and 6.

[0312] In some embodiments, the purified recombinant protein is a monoclonal antibody. In some embodiments, the monoclonal antibody specifically binds to an epitope of CD20 (also referred to herein as an "anti-CD20 antibody"). In some embodiments, the anti-CD20 antibody specifically binds to the same epitope as TG-1101.

[0313] In some embodiments, the biological properties of the anti-CD20 antibodies made by the methods disclosed herein can be measured and described in assays using comparison to a reference standard. In some embodiments, the reference standard is a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808. RS-117808 (“antibody ublituximab (TG-1101)”) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, in accordance with the terms of the Budapest Treaty, which was received by the ATCC on April 15, 2022 and assigned the unofficial Patent Deposit Number PTA-127294.

[0314] In some embodiments, the reference standard is an anti-CD20 antibody. In certain embodiments, the reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab).

[0315] In some embodiments, the monoclonal antibodies are subjected to one or more binding assays 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 embodiments, the recombinant antibodies are assayed for antibody-dependent cellular cytotoxicity (ADCC) biological activity / potency and / or complement-dependent cytotoxicity (CDC) biological activity / potency as described in Examples 1 and 2, etc. Additional assays for assaying the quality or function of a protein or antibody are well known to those of skill in the art.

[0316] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 82% to 138% in a cell-based CD20 binding activity bioassay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 92% to 118% in a cell-based CD20 binding activity bioassay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 109% in a cell-based CD20 binding activity bioassay compared to that of a commercial reference standard. In some embodiments, the percentage of CD20 binding is determined by a cell-based CD20 binding activity bioassay, such as the binding of an anti-CD20 antibody to the CD20-expressing human mantle cell lymphoma cell line, Jeko-1.

[0317] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 82% to 130% for FcγRIIIa-158V binding compared to a commercial reference standard when measured by surface plasmon resonance (SPR). In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 76% to 130% for FcγRIIIa-158V binding compared to a commercial reference standard when measured by surface plasmon resonance (SPR).

[0318] In some embodiments, the monoclonal antibody has a KD value of 30 - 70 nM in the FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In some embodiments, the monoclonal antibody has a KD value of about 59 nM in the FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In some embodiments, the monoclonal antibody has a KD value of 500 - 1000 nM in the FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In some embodiments, the monoclonal antibody has a KD value of 760 nM in the FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In some embodiments, the monoclonal antibody has a binding affinity for FcγRIIIa 158V or FcγRIIIa 158F that is significantly higher than that of the anti-CD20 antibody rituximab.

[0319] In some embodiments, the monoclonal antibody produced by the methods disclosed herein has a relative potency of 86 - 117% in the C1q binding assay as measured by ELISA, compared to a commercial reference standard. In some embodiments, the monoclonal antibody produced by the methods disclosed herein has a relative potency of 86% - 116% in the C1q binding assay as measured by ELISA, compared to a commercial reference standard. In some embodiments, the monoclonal antibody produced by the methods disclosed herein has a relative potency of 88 - 113% in the C1q binding assay as measured by ELISA, compared to a commercial reference standard. In some embodiments, the monoclonal antibody produced by the methods disclosed herein has a relative potency of about 99% in the C1q binding assay as measured by ELISA, compared to a commercial reference standard.

[0320] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a higher percentage of antibody-dependent cellular cytotoxicity (ADCC) activity compared to monoclonal antibodies produced by a culture process that does not use culture conditions of 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.

[0321] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein induce greater cytotoxicity in a cell-based antibody-dependent cellular cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, belimumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0322] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of about 117% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the cell-based ADCC assay uses effector cells selected from CD16 effector cells and primary NK cells. In some embodiments, the population performs cell-based ADCC using CD16 effector cells that is greater than 100% of that of the commercial reference standard.

[0323] In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 74% to 127% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 73% to 128% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of 78 to 116% in a cell-based CDC assay compared to that of a commercial reference standard. In some embodiments, the monoclonal antibodies produced by the methods disclosed herein have a relative potency of approximately 91% in a cell-based CDC assay compared to that of a commercial reference standard.

[0324] In some embodiments, a monoclonal antibody produced by any of the methods disclosed herein comprises a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0325] In some embodiments, a monoclonal antibody comprises a heavy chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7, and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0326] In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having the amino acid sequence set forth in SEQ ID NO: 9. 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.

[0327] In some embodiments, the methods disclosed herein yield a recombinant protein (e.g., anti-CD20 antibody) harvest titer of about 0.5 g / L to about 1.5 g / L. In some embodiments, the harvest titer is from about 1.0 g / L to about 1.5 g / L.

[0328] In some embodiments, the anti-CD20 antibody is TG-1101 or an antibody that binds to the same epitope as TG-1101. In some embodiments, the anti-CD20 antibody is TG-1101.

[0329] Commercial scale production of recombinant proteins in rat hybridoma cells Also provided is commercial scale (e.g., 10,000 L to 25,000 L) production of a recombinant protein (e.g., anti-CD20 antibody) expressed in rat hybridoma cells. See Example 3.

[0330] In some embodiments, a) preparing and thawing a working rat hybridoma cell bank of the recombinant protein of interest; and b) culturing the rat hybridoma cells obtained from the cell bank in a series of shake flasks (125 mL, 500 mL, 3 L, 3 x 3 L shake flasks and 50 L cell bags) with respect to size and volume, by (though), at least 0.30 x 10 6increasing by the target seeding density of cells / mL; c) treating the cell culture with a series of seed bioreactors (120 L, 600 L and 3,000 L) to further increase the volume and cell culture mass; d) inoculating the cell culture from the 3,000 L seed bioreactor into a 15,000 L production bioreactor; e) collecting the cell culture supernatant from the 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; h) inactivating viral agents by solvent-detergent virus inactivation (SDVI). A method for producing a recombinant protein in the culture of rat hybridoma cells on a commercial scale is provided, which comprises the steps of:

[0331] In some embodiments, protein A column chromatography is performed to purify the recovered recombinant protein, reduce process impurities such as cell culture components, HCP and residual DNA, and provide virus safety. In some embodiments, the protein A column chromatography is performed using MabSuRe Select resin (Cytiva) in a bind / elute mode.

[0332] In some embodiments, the packed column is assayed for HETP performance using a sodium acetate / benzyl alcohol buffer. In some embodiments, column operation is performed at 13 - 25°C. In some embodiments, prior to loading, the column is sanitized with 0.5 M sodium hydroxide, flowed with WFI, and equilibrated with an equilibration buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1). In some embodiments, the clarified harvest is mixed briefly and then loaded onto the column using a maximum of 21 g recombinant protein / L resin load. The column is washed with Wash Buffer 1 (equilibration buffer), followed by a second wash with Wash Buffer 2 of high salt concentration (25 mM Tris, 1.2 M NaCl, 5 mM EDTA, pH 7.1), and then an additional wash using Wash Buffer 3 (equilibration buffer). In some embodiments, the clarified harvest is loaded onto the column using a maximum of 36 g recombinant protein / L resin load. The bound recombinant protein is eluted at 200 - 220 cm / h with an elution buffer (25 mM sodium citrate, pH 3.6) using elution peak collection by A280 such that it does not exceed 1.2 column volumes. In some embodiments, the bound recombinant protein is eluted at 200 - 220 cm / h with an elution buffer (25 mM sodium citrate, pH 3.6) using elution peak collection by A280 such that it does not exceed 2.4 column volumes. The eluate is collected in a tank containing a neutralization buffer (2.0 M Tris, pH 7.5) and filtered through a 0.2 μm filter before transferring to a different tank. The Protein A column is sanitized with 0.5 M sodium hydroxide. Up to 3 cycles per batch can be run; if multiple cycles are required for the Protein A process, the column is re-equilibrated with the equilibration buffer for the next cycle. After sanitization, the Protein A column is neutralized with the equilibration buffer and stored at 13 - 25°C in 200 mM sodium acetate, 2% benzyl alcohol, pH 5.0. The pooled (if 2 or more cycles) and neutralized eluate is diluted to a concentration of ≤10 g / L with 5 mM sodium phosphate, pH 7.2 and stored at 13 - 25°C for ≤72 hours or at 2 - 8°C for ≤11 days.In some embodiments, the pooled (in the case of 2 or more cycles), neutralized eluate is diluted to a concentration of ≤10 g / L with 5 mM sodium phosphate, pH 7.2 and stored at 13 - 25 °C for ≤24 hours.

[0333] In some embodiments, following the Protein A capture chromatography step, a solvent detergent virus inactivation (SDVI) step is performed to inactivate potential viral agents. In some embodiments, the Protein A elution pool is diluted and treated with 3.5% (v / v) TnBP, 12% (w / v) polysorbate 80 and held at 24.0 - 26.0 °C for at least 120 minutes while mixing. The SDVI pool is filtered through a 0.2 μm filter and then transferred to a different tank where it is diluted to 50 mOsm / kg with 5 mM sodium phosphate, pH 7.2 and the pH is adjusted to 7.2 if necessary. After pH adjustment, the pool is held at 13 - 25 °C for ≤30 hours before proceeding to the CEX column. In some embodiments, the pool is held at 13 - 25 °C for ≤24 hours.

[0334] In some embodiments, the method further comprises purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX).

[0335] In some embodiments, further purification of the recombinant protein and removal of residual process impurities are performed using cation exchange column chromatography (CEX). In some embodiments, CEX was performed using SP Sepharose Fast Flow (Cytiva) in a bind / elute mode. In some embodiments, the packed column was assayed for HETP performance using a buffer containing sodium acetate / benzyl alcohol, and all column manipulations were performed at 13-25°C. In some embodiments, the column was sanitized with 0.5 M sodium hydroxide, rinsed with WFI, and equilibrated with an equilibration buffer (20 mM sodium phosphate, pH 7.2). The virus inactivation / dilution solution was loaded onto the column at a maximum resin load of 65 g / L. The column was washed with Wash Buffer 1 (equilibration buffer), followed by a second wash with Wash Buffer 2 (equilibration buffer in the reverse direction). The bound recombinant protein was eluted using elution peak collection with 20 mM sodium phosphate, 150 mM NaCl, pH 7.2 and A280 monitoring. In some embodiments, the eluate was filtered (0.2 μm) and stored at 13-25°C for ≤72 hours or at 2-8°C for ≤11 days. In some embodiments, the eluate was stored at 13-25°C for ≤24 hours. After elution, the column was stripped with 2 M NaCl, followed by sanitization with 0.5 M sodium hydroxide. One cycle per batch was possible. After completion, the column was sanitized (0.5 M sodium hydroxide) and stored in a storage buffer (200 mM sodium acetate, 2% benzyl alcohol, pH 5.0).

[0336] In some embodiments, further purification of the recombinant protein is performed using anion exchange membrane chromatography. In some embodiments, AEX is performed using a Mustang Q (Pall Corporation) membrane absorber (MA) filter in flow-through mode. In some embodiments, the membranes are disposable (i.e., individual membranes cannot be reused), and several membrane capsules can be used at an appropriate load level per batch. In some embodiments, the eluate obtained from the CEX step is diluted with 20 mM sodium phosphate, pH 8.0, followed by adjustment to pH 8.0. The number of cycles is calculated based on the protein concentration such that the load is 200 - 700 g recombinant protein / L membrane load. The membranes are sanitized with 0.5 M sodium hydroxide, flowed with 2 M NaCl then WFI, and then equilibrated with equilibration buffer (20 mM sodium phosphate, 75 mM NaCl, pH 8.0) in preparation for loading. After loading, the membranes are chased with 20 mM sodium phosphate, 75 mM NaCl, pH 8.0 to maximize recovery. The collected flow-through containing the product from all cycles is 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 is adjusted to 6.8. In some embodiments, the adjusted AEX pool is stored at 13 - 25°C for ≤72 hours or at 2 - 8°C for ≤11 days. In some embodiments, the adjusted AEX pool is stored at 13 - 25°C for ≤24 hours.

[0337] In some embodiments, the method further includes performing viral filtration (VF) to remove potential viruses including small viruses such as parvovirus. In some embodiments, VF is performed by filtering through a Viresolve prefilter in a series by a Viresolve Pro virus filter (Millipore Sigma) at an operating temperature target range of 13 - 25°C. The process used a filter sufficient to meet the load limit. To perform the filtration, the filters were installed in series, flowed with WFI, integrity tested, and then sterilized with 0.5 M sodium hydroxide. This was followed by flowing with an equilibration buffer (25 mM sodium citrate, 154 mM NaCl, pH 6.5). The filtered Mustang Q membrane flow-through was processed through a virus reduction filter.

[0338] The protein concentration was determined and used to confirm that the membrane load ratio was ≦600 g / m 2 After loading, the membrane was chased with equilibration buffer and an integrity test after use was performed. In some embodiments, the virus filtrate was stored at 13 - 25°C for ≦72 hours or at 2 - 8°C for ≦11 days. In some embodiments, the virus filtrate was stored at 13 - 25°C for ≦33 hours.

[0339] In some embodiments, commercial-scale production is carried out in a 10,000 L - 25,000 L production bioreactor. In some embodiments, commercial-scale production is carried out in a 15,000 L production bioreactor. In some embodiments, commercial-scale production is operated in a fed-batch mode.

[0340] In some embodiments, a drill hole (10) gas sparger having a 4.0 mm orifice diameter is used in a 15,000 L production bioreactor.

[0341] In some embodiments, the method further includes ultrafiltration / diafiltration (UFDF).

[0342] In some embodiments, the method further comprises preparing a bulk drug substance formulation comprising a recombinant protein, including adding polysorbate 80 in a formulation buffer to prepare the bulk drug substance formulation. In some embodiments, the method further comprises subjecting the bulk drug substance formulation to 0.2 μm filtration.

[0343] In some embodiments, the method further comprises 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.

[0344] In some embodiments, the recombinant protein in the bulk drug substance formulation is formulated into a pharmaceutically acceptable formulation.

[0345] In some embodiments, the method further comprises testing an unprocessed bulk harvest from a 15,000 L production bioreactor for microbial and viral adventitious agents and removing microbial and viral adventitious agents from the 15,000 L production bioreactor.

[0346] In some embodiments, the rat hybridoma cells are YB2 / 0 cells.

[0347] In some embodiments, the recombinant protein produced by the manufacturing method is a monoclonal antibody. In some embodiments, the monoclonal antibody binds to an epitope of CD20 (i.e., is an anti-CD20 antibody). In some embodiments, the monoclonal antibody comprises a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] In some embodiments, the monoclonal antibody is TG-1101, or an antibody that binds to the same epitope as TG-1101. In some embodiments, the monoclonal antibody is TG-1101. As described below, in some embodiments, monoclonal antibodies produced by the methods disclosed herein contain a unique glycosylation signature.

[0352] Rat Hybridoma Master Cell Bank and Working Cell Bank 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 (e.g., monoclonal antibodies) disclosed herein. See Example 3. The MCBs and WCBs disclosed herein were phenotypically stable over 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, based on, for example, cell growth, harvest titer, productivity and product quality. In some embodiments, the MCBs and WCBs disclosed herein were phenotypically stable over up to 71 cell generations, based on, for example, cell growth, harvest titer, productivity and product quality. Ibid.

[0353] In some embodiments, the rat hybridoma MCB provided herein comprises a recombinant protein 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) a peak viable cell density of about 11 to about 13×106 cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a percent fucosylation of about 30% to about 38%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimer as detected by SEC; vi) undetectable to about 3% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaging 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.

[0354] In some embodiments, the rat hybridoma MCB provided herein comprises a recombinant protein 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) a peak viable cell density of about 11 to about 13×106 cells / mL; ii) a harvest titer of about 650 to about 720 mg / L; iii) a percent fucosylation of about 30% to about 38%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1.5% to about 2% dimer as detected by SEC; vi) undetectable to about 3% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 25% to about 30% acidic isoforms as detected by imaging 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.

[0355] In some embodiments, the rat hybridoma WCB provided herein comprises a recombinant protein having at least two of the following parameters: i) a peak viable cell density of about 11 to about 28×106 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a percent fucosylation of about 18% to about 40%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimer as detected by SEC; vi) undetectable to about 2% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaging 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.

[0356] In some embodiments, the rat hybridoma WCB provided herein comprises a recombinant protein having at least two of the following parameters: i) a peak viable cell density of about 11 to about 28×106 cells / mL; ii) a harvest titer of about 420 to about 1280 mg / L; iii) a percent fucosylation of about 18% to about 40%; iv) about 97% to about 99% monomer as detected by size exclusion chromatography (SEC); v) about 1% to about 2% dimer as detected by SEC; vi) undetectable to about 2% level of aggregates as detected by SEC; vii) undetectable to about 1% level of fragments as detected by SEC; viii) about 19% to about 31% acidic isoforms as detected by imaging 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.

[0357] In some embodiments, the rat hybridoma cells in the MCB composition or the 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.

[0358] In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth 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 set forth in SEQ ID NO: 7; and a light chain having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain having the amino acid sequence set forth in SEQ ID NO: 9.

[0359] Also provided is a method of making a recombinant protein by using the MCB or WCB compositions disclosed herein. In some embodiments, 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 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.

[0360] Recombinant Proteins and Compositions Provided are recombinant proteins made according to any of the methods disclosed herein. In some embodiments, the recombinant protein is a monoclonal antibody. In some embodiments, the monoclonal antibody is an antibody that binds to an epitope of CD20. In some embodiments, the monoclonal antibody is an antibody that binds to the same epitope as TG-1101 (TG Therapeutics, Inc.). In some embodiments, the monoclonal antibody is TG-1101.

[0361] In one aspect, the present disclosure provides a composition comprising a recombinant protein produced in rat hybridoma cells (e.g., YB2 / 0) by any of the methods disclosed herein. In some aspects, the recombinant protein is a monoclonal antibody. In some aspects, the monoclonal antibody targets the CD20 antigen (i.e., is 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.

[0362] In some aspects, a composition produced by the methods disclosed herein (e.g., comprising an anti-CD20 antibody) has a unique glycosylation / N-glycan profile characterized by, for example, between about 20% and about 40% fucosylated glycan and / or between about 10% and about 20% galactosylated glycan.

[0363] In some aspects, the monoclonal antibody comprises an N-glycan profile that includes one or both of the following: i. about 10-20% galactosylated glycan; and / or ii. about 23-36% fucosylated glycan.

[0364] In some aspects, the monoclonal antibody comprises an N-glycan profile that includes about 10-20% galactosylated glycan and about 23-36% fucosylated glycan.

[0365] In some aspects, the monoclonal antibody comprises an N-glycan profile that includes about 36% fucosylated glycan.

[0366] In some aspects, the monoclonal antibody comprises an N-glycan profile that includes about 16-18% galactosylated glycan.

[0367] In some aspects, the N-glycan profile includes about 17% galactosylated glycan.

[0368] In some embodiments, the monoclonal antibody comprises an N-glycan profile that includes at least about 10% bisecting N-glycan. In some embodiments, the N-glycan profile includes from about 12% to 30% bisecting N-glycan. In some embodiments, the N-glycan profile includes about 18% bisecting N-glycan.

[0369] In some embodiments, the monoclonal antibody comprises an N-glycan profile that includes less than 5% sialylated glycan. In some embodiments, the N-glycan profile includes less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycan. In some embodiments, the N-glycan profile does not include a detectable amount of sialylated glycan.

[0370] In some embodiments, the monoclonal antibody comprises an N-glycan profile that includes from 0.1% to 1.5% Man5 N-glycan. In some embodiments, the N-glycan profile includes from 0.4% to 0.7% Man5 N-glycan. In some embodiments, the N-glycan profile includes about 0.6% Man5 N-glycan. In some embodiments, Man5 N-glycan is the only high-mannose species in the N-glycan profile.

[0371] In some embodiments, a cell culture composition comprising a recombinant protein, monoclonal antibody, anti-CD20 antibody or TG-1101 produced in rat hybridoma cells by any of the methods disclosed herein is a pharmaceutical composition, and the recombinant protein, monoclonal antibody, anti-CD20 antibody or TG-1101 is formulated with a pharmaceutically acceptable carrier.

[0372] As used herein, the phrase "pharmaceutical composition" refers to a composition that is acceptable for administration of a medicament to humans and the like. Such a composition can contain substances that are impurities at levels that do not exceed levels acceptable for administration of a medicament (such levels include the absence of such impurities), and for example, in formulating such a composition to facilitate administration, in addition to any active agent(s), can contain pharmaceutically acceptable excipients, vehicles, carriers, and other inert components.

[0373] In some embodiments, the pharmaceutical composition contains one or more of the following: sodium chloride, trisodium citrate anhydrous, polysorbate 80, and hydrochloric acid.

[0374] Also provided is a method of treating a hematological malignancy in a subject in need thereof by administering a recombinant protein or monoclonal antibody (e.g., an anti-CD20 antibody) made according to any of the methods disclosed herein. In some embodiments, the hematological cancer is lymphoma, leukemia, or myeloma. In some embodiments, the hematological cancer is selected from B-cell lymphoma, acute lymphoblastic 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), Waldenström 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).

[0375] Also provided is a method of treating an autoimmune disorder in a subject in need thereof by administering a recombinant protein or monoclonal antibody (e.g., an anti-CD20 antibody) made according to any of the methods disclosed herein. In some embodiments, 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), graft rejection, graft-versus-host disease, lupus erythematosus, inflammatory disease, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory disease or uveitis.

[0376] In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the multiple sclerosis is relapsing multiple sclerosis. In certain embodiments, the relapsing multiple sclerosis is clinically isolated syndrome (CIS); relapsing-remitting MS (RRMS); active secondary progressive MS (SPMS); or primary progressive MS (PPMS). In a preferred embodiment, the subject is human.

[0377] The following examples are provided by way of illustration and not limitation.

Examples

[0378] In the following examples, a cell culture process for controlling the growth and productivity of an anti-CD20 IgG1 monoclonal antibody (TG-1101) expressed by YB2 / 0 rat hybridoma cells is illustrated, for example, by the selection of cell culture medium (Example 1), culture pH, temperature, and pCO2 control methodologies (Example 2). Example 3 illustrates the 15,000 L commercial scale production of anti-CD20 antibody in rat hybridoma cells. Example 4 illustrates the detection and removal of viruses or other adventitious agents in the 15,000 commercial scale production process. Example 5 illustrates the calculated pH process parameter, "integrated pH2 difference", and its effect on the harvest titer, percent fucosylation, and integrated viable cell density (IVCD). Finally, Example 6 illustrates the unique glycosylation profile of the anti-CD20 antibody produced by the methods disclosed herein.

[0379] Protein fucosylation levels were also decreased and antibody Fc effector activity was also increased while inducing an increase in process productivity. A demonstrated process for controlling both productivity and product quality is shown using this unique, generally non-utilized mammalian expression system. The observed results are 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)), it has not been shown that using such cells, the fucosylation level can be directed lower and the productivity can be directed higher by cell culture process means alone. Furthermore, an anti-CD20 antibody having a unique glycosylation signature and, needless to say, the production of anti-CD20 antibody in rat hybridoma cells at commercial scale has not been demonstrated.

[0380] [Example 1] Increase in anti-CD20 antibody productivity expressed by YB2 / 0 rat hybridoma cell line by selection of cell culture medium Materials and Methods Cell Culture The YB2 / 0 rat hybridoma cell line (ATCC CRL 1662) was adapted to 10 commercially available cell culture media (Table 2). After thawing and scaling up in EM-SF2-P5-H4 medium in a static T-flask, the cells were subcultured into 125 mL shake flasks containing each of the 10 new media (at 2 - 8 °C), and placed in a humidified incubator at 37 °C with stirring at 90 RPM with %CO2 corresponding to the manufacturer's recommendation for each medium. The cell cultures were subcultured daily at a seeding density of 0.5×10 6 cells until the cell viability dropped below 60% (in which case the culture was terminated), or until the growth rate viability reached a steady state over 5 days (after which the cells were considered adapted). To determine the optimal subculture strategy, growth curves were performed in the medium with the best performance. All media were supplemented with 6 mM L-glutamine together with 1 mL / L of 1000× cholesterol.

[0381]

Table 3

[0382] After the medium adaptation period in 125 mL shake flasks, the cells were determined to have successfully adapted to four new media: CDM4Mab, CD Hybridoma, Hycell, and ProDoma 1. To obtain seven unique basal medium conditions for further screening against six commercially available feeds (Table 3), the top-performing medium (CDM4Mab®) in terms of growth and viability was blended with the other three successful media at a 50:50 ratio. Using a definitive screening design of experiments (DOE), the top-performing combinations of growth and feed media were identified when runs were conducted in fed-batch mode using the Ambr15 cell culture platform (Sartorius). Additionally, one vessel was run using historical basal medium and feed as a control, and five vessels were run using the top-performing new medium (CDM4Mab®) supplemented with both varying concentrations of fatty acid supplement and Long R3 IGF-1 supplement. The process parameters used for all vessels are shown in Table 4. The measured outputs for each parameter were the viable cell density, cell viability, antibody titer, and glycosylation profile of the expressed mAb at the collection date for the top 12 conditions (including the control).

[0383]

Table 4

[0384]

Table 5

[0385] Protein Analytical Characterization N-Glycan N-linked glycans are cleaved from the products by enzymatic deglycosylation using PNGase F and labeled with the fluorescent compound 2-aminobenzamide (2-AB). The labeled glycans are developed using a hydrophilic interaction partition mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360 nm and emission at 428 nm). 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 chromatograms is based on retention time and is identified relative to the peaks in glycan standards confirmed by mass spectrometry.

[0386] ADCC Potency Assay The ADCC assay is a cell-based assay using Eurofins-DiscoverX "KILR CD16a effector cells", which are CD20-expressing Jeko-1 cells, a human mantle cell lymphoma cell line, and single-donor-derived human CD8+ T-lymphocytes engineered to express CD16 (FcγRIII) on their plasma membrane surfaces. Target cell lysis mediated by the antibody sample is measured. The dose-response curve is modeled using 4-parameter logistic regression (4PL), and the results are reported as % potency compared to a reference standard.

[0387] CDC Potency Assay The CDC assay is a cell-based assay using CD20-expressing Jeko-1 cells 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 compared to a reference standard.

[0388] CD20 Binding Activity In the CD20 binding activity assay, the binding of anti-CD20 antibodies to the CD20-expressing human mantle cell lymphoma cell line, Jeko-1, is evaluated using electrochemiluminescence (Meso Scale Discovery). The dose-response curve is modeled using a 4PL equation and the results are reported as % potency compared to a reference standard.

[0389] FcγRIIIa binding The binding of anti-CD20 antibody samples to FcγRIIIa is evaluated by surface plasmon resonance (SPR). This method follows a direct binding assay methodology, according to which the Fc receptor (ligand) is immobilized directly onto the appropriate flow cell on the sensor chip surface and the analyte is injected over the chip to assess binding. The high-affinity allele of FcγRIIIa (158V) is determined.

[0390] The equilibrium dissociation constant (KD) and relative affinity of each sample compared to the reference standard are determined for each receptor. The rate of change of the SPR signal is analyzed using a 1:1 Langmuir model to obtain the apparent rate constants for the association and dissociation phases of the reaction, as well as the equilibrium dissociation constant. The results are reported as % potency compared to the reference standard for the FcγRIIIa-158V variant.

[0391] C1q binding The binding of anti-CD20 antibodies to C1q is evaluated using a method based on C1q ELISA. The dose-response binding is modeled by weighted non-linear regression using a 4-parameter logistic fit and the results are reported as % potency compared to a reference standard.

[0392] Results After successfully adapting the cells to the new medium, the adapted cells were used to inoculate an Ambr 15 bioreactor (Sartorius). The objective of this study was to identify a combination of growth medium and feed medium that would result in approximately twice the titer and similar product quality as a fed-batch process using PROEMS-02 growth medium and Cell Boost 3 feed medium (approximately 320 mg / L, 40 - 60% fucosylated). This study was carried out using the process conditions shown in the method above.

[0393] A wide range of cell growth performance was observed across different combinations of growth medium and feed medium (Table 5), and the integrated viable cell density (IVCD) results on day 10 ranged from 10 - 87×10 6 viable cells / mL / day (Figure 2), and the cell viability on day 10 ranged from 7.5 - 93.5% (Figure 3). Figure 4 depicts the top 11 medium conditions from the perspective of the harvest titer (plus control condition: PRO-EMS-02 / Cell Boost 3), and Figure 5 depicts the percent (%) fucosylation results of the top 11 medium conditions plus control. All 11 conditions were superior in performance to the control medium from the perspective of titer, and CDM4Mab was the best represented among the top 11 new combinations of growth medium and feed medium. The combination of BalanCD CHO Feed 4 and CDM4Mab medium produced the highest titer of 0.563 g / L and represents the only medium / feed combination approaching a two-fold increase in titer.

[0394] From a product quality perspective, CDM4Mab / BalanCD CHO Feed 4 demonstrated significantly lower % fucosylation (approximately 24%) compared to the range supported by control conditions (i.e., 40 - 60%). Additionally, when the CDM4Mab growth medium was blended with the CD hybridoma medium at a 50:50 ratio and combined with the feed medium BalanCD CHO Feed 4, the results were still top-performing conditions from the perspective of titer (approximately 0.38 g / L), but the overall % fucosylation increased to approximately 72%. This result suggests that by varying the ratio of different growth media while maintaining a consistent feed medium (BalanCD CHO Feed 4), the % fucosylation results can be varied in mAbs.

[0395]

Table 6-1

[0396]

Table 6-2

[0397] The cell culture growth and feed medium identified in the above study were then used in the clinical GMP manufacture of TG-1101, an anti-CD20 IgG1 monoclonal antibody. The resulting drug substance was examined for % fucosylation and Fc effector function / activity. Comparison of the mAb characterization results produced using the control media (PROEMS-02 growth medium and Cell Boost 3 feed medium) vs. the new media (CDM4Mab® growth medium and BalanCD CHO Feed 4® feed medium) are shown in Figure 6 for % fucosylation, Figure 7 for FcγRIIIa-158V binding, Figure 8 for CD20 binding, Figure 9 for ADCC activity, Figure 10 for C1q binding, and Figure 11 for CDC activity. The results show a significant drop in % fucosylation, accompanied by a significant increase in FcγRIIIa-158V binding and a moderate increase in ADCC activity. There was no change in CD20 antigen binding as a result of the cell culture media switch. In addition, there was no change in C1q binding and little to no change in CDC activity. Overall, these results suggest that by changing the cell culture medium for this rarely used YB2 / 0 rat hybridoma cell line, the function and activity of the expressed antibody can be modulated by cell culture media means alone. There is no literature reporting this relationship in YB2 / 0 rat hybridoma cells in the public domain.

[0398] Conclusion In this example, one way to increase process performance (cell growth, recombinant protein productivity) and modulate protein glycosylation from a cell culture process using YB2 / 0 cells was demonstrated. In this example, the cell growth and feed medium were found to have a significant role in directing the protein glycosylation profile. Further examination identified a combination of growth and feed media (CDM4Mab®, BalanCD CHO Feed 4®) that supports a significant decrease in the percent fucosylation level, which in turn supports an increase in potency via FcγRIIIa binding.

[0399] [Example 2] Increase in anti-CD20 antibody productivity by YB2 / 0 rat hybridoma cell line by cell culture pH, temperature and pCO2 control methodology Materials and methods Cell culture 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 at 37 °C in a growth medium of known composition, CDM4Mab (GE Healthcare). After a series of passages into increasing volume culture vessels and after sufficient cell biomass had been achieved, the cells were inoculated into a 10 L laboratory-scale bioreactor. The process conditions utilized in these cultures are shown in Table 6.

[0400] The 10 L bioreactor was equilibrated after addition of the cell growth medium. The medium was added to the initial volume target. The dissolved oxygen (DO) and pH probes were calibrated before use. The initial bioreactor set points included temperature, pH, dissolved oxygen and agitation speed. The bioreactor was inoculated with a cell culture from a seed train inoculum vessel at a viable cell density target of 0.5 × 106 viable cells / mL. In the production bioreactor process, the pH was controlled by base addition and CO2 injection as required. The dissolved oxygen was controlled by oxygen and air injection as required. Antifoam was added if necessary to reduce concerns of foaming. Offline pH, pCO2 / pO2, weight osmolarity and metabolites were monitored daily using a blood gas analyzer (Siemens), as well as viable cell density (VCD) and viability using a ViCell automated cell counter (Beckman Coulter). Daily samples were also measured for weight osmolarity using a Nova Bioprofile FLEX2 analyzer (Nova Biomedical), along with process metabolites including glucose, lactate, glutamine, ammonia.

[0401] Throughout the bioreactor culture process, various feed media and solutions were added to support the nutritional requirements of the cells. BalanCD CHO Feed4® medium (Irvine Scientific) was added on days 3, 5, 7, and 9 at a specified volume (4.0% of the initial bioreactor working volume). When the daily bioreactor sample measurements were < 3.0 g / L on the specified process days, a calculated volume of concentrated glucose solution was provided (up to 4.0 g / L). A fixed volume of glutamine solution was provided as a bolus on day 3, and when the daily bioreactor sample measurements were < 3.00 mM on the specified process days, the calculated volume was provided (up to 4.0 mM). On days 0 and 4, a 1000× concentrated cholesterol lipid solution was added to the culture in a fixed bolus addition (0.256% v / v).

[0402] Throughout the bioreactor culture process, the process set points were shifted at fixed times. The culture pH set point was shifted from 7.10 + / - 0.30 on process day 3 to different pH set points (Table 6). The temperature set point was shifted from 37 °C to 35 °C on process day 1 and then shifted again from 35 °C to 32.5 °C on process day 3.

[0403] The production bioreactor was harvested based on either the culture duration or the earlier occurrence of the cell viability criteria. For product quality measurements (SEC, iCIEF, N-linked glycan) and functional assays including both effector function and binding assays, the cell culture harvest was clarified to remove cells and then Protein A purification was performed.

[0404]

Table 7

[0405] Protein Analytical Characterization N-Glycan N-linked glycans are cleaved from the products by enzymatic deglycosylation using PNGase F and labeled with the fluorescent compound 2-aminobenzamide (2-AB). The labeled glycans are developed using a hydrophilic interaction partition mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360 nm and emission at 428 nm). 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 peaks in the glycan standards confirmed by mass spectrometry.

[0406] SEC The SEC-HPLC method is used to determine the molecular size distribution and purity of IgG1 antibodies. SE-HPLC separates proteins based on hydrodynamic radius and uses a TSKgel G3000SWXL column (Tosoh) designed to separate proteins in the range of 10,000 - 500,000 Da. The resulting chromatogram is monitored at 214 nm, peaks are integrated, and the amount of each peak (monomer, dimer, aggregate, and fragment) is expressed as a percentage of its area. Sample, reference standard, molecular weight standard, and blank injections are performed to assess system suitability.

[0407] iCIEF Imaged capillary isoelectric focusing (iCIEF) was used to evaluate and quantify the distribution of charge-based isoforms of TG-1101 using a Protein Simple iCE3 analyzer. The test sample solution contained 0.15% methyl cellulose, 1.2 M urea, 8% ampholytes pH 3-10.5 and pH 10.10 and 8.40 pH markers. Focusing was carried out at 1500 V for 1 minute followed by 3000 V for 8 minutes. The results reported include the pI of the major peak, the % peak area of the major peak, and the pooled values of the % peak areas of the basic and acidic peaks. Blank samples and hemoglobin controls were also run to evaluate system suitability.

[0408] ADCC Potency Assay The ADCC assay is a cell-based assay using Eurofins-DiscoverX "KILR CD16a effector cells", which are CD20-expressing Jeko-1 cells, a human mantle cell lymphoma cell line, and single donor-derived human CD8+ T-lymphocytes engineered to express CD16 (FcγRIII) on their plasma membrane surface. Target cell lysis mediated by the antibody sample is measured. The dose-response curve is modeled using a 4PL equation and the results are reported as % potency compared to a reference standard.

[0409] CDC Potency Assay The CDC assay is a cell-based assay using CD20-expressing Jeko-1 cells and rabbit serum as a source of complement. CDC-mediated cell lysis is measured. The dose-response curve is modeled using a 4PL equation and the results are reported as % potency compared to a reference standard.

[0410] CD20 Binding Activity In the CD20 binding activity assay, the binding of anti-CD20 antibody to CD20-expressing human mantle cell lymphoma cell line, Jeko-1, is evaluated using the Meso Scale Discovery (MSD) method. The dose-response curve is modeled using a 4PL equation and the results are reported as % potency compared to a reference standard.

[0411] FcγRIIIa-158V Binding: The binding of anti-CD20 antibody samples to FcγRIIIa is evaluated by surface plasmon resonance (SPR). This method follows a direct binding assay methodology, according to which the Fc receptor (ligand) is directly immobilized onto a suitable flow cell on the sensor chip surface, and the analyte is injected over the chip to assay for binding. The high-affinity allele of FcγRIIIa (158V) is determined.

[0412] The equilibrium dissociation constant (KD) and relative affinity of each sample compared to the reference standard are determined for each receptor. The rate of change of the SPR signal is analyzed using a 1:1 Langmuir model to obtain the apparent rate constants for the association and dissociation phases of the reaction, as well as the equilibrium dissociation constant. Results are reported as % potency compared to the reference standard for the FcγRIIIa-158V variant.

[0413] C1q Binding: The binding of anti-CD20 antibody to C1q is evaluated using a method based on C1q ELISA. The dose-response binding is modeled by weighted non-linear regression using a 4-parameter logistic fit, and results are reported as % potency compared to the reference standard.

[0414] Results Manufacture of the YB2 / 0 rat hybridoma cell line and retrospective analysis of laboratory-scale cell culture were 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 (Figure 12). Generally, cultures with cumulative cell growth (IVCD) also had lower cumulative exposure to pCO2 (integrated pCO2). Another significant trend in the data was the positive relationship between harvest titer and cumulative culture exposure to pCO2 (Figure 13). Generally, cultures with high harvest titers of monoclonal antibody also had lower integrated pCO2. Further investigation of these relationships was pursued. To facilitate changes in pCO2, the use of different pH control strategies was specifically examined to evaluate 10 L laboratory-scale cultures.

[0415] Production bioreactor cultures were performed at laboratory scale by evaluating different post-shift pH setpoints and control ranges. The cell growth results are shown in Figure 14, demonstrating the pH-dependent effect on the observed results. At the lowest post-shift pH control range studied (6.70 + / - 0.10), the peak viable cell density (VCD) was the lowest among the conditions examined (18.9 x 106 cells / mL). In contrast, at the highest pH control range studied (6.95 + / - 0.05), the peak VCD was the highest among the conditions examined. The pH control ranges between these two ranges demonstrated intermediate cell growth and peak VCD results. The results emphasize that a significant increase in cell growth was observed as the process pH control range increased. Previously, lower cell growth due to lower culture pH was observed in CHO cells (Trummer, E. et al., Biotechnology and Bioengineering 94:1033-1044 (2006)). However, this behavior has not been reported for the YB2 / 0 cell line.

[0416] Cell viability results also showed trends in different pH control ranges, as shown in Figure 15. In the lowest pH control range studied, cell viability dropped more quickly, inducing bioreactor harvest on day 13 of the process. In the higher pH control ranges studied, cell viability dropped much more slowly, resulting in bioreactor harvest on day 14 of the process. These results are significant as they suggest that pH-only control for this particular cell line is a process means that can increase culture lifespan. Along with such an extended culture lifespan, the resulting impact on the harvest titer is also significant.

[0417] Glucose is consumed by mammalian cells as the primary source of energy and carbon required to perform oxidative metabolism. The major waste byproduct from glucose metabolism in mammalian cells is lactate. High levels of lactate have previously been shown to be inhibitory to process performance (Hassell, T. et al., Appl Biochem Biotechnol. 30: 29-41 (1991)). Glucose and lactate levels in the cell culture medium were monitored throughout each of the cultures (Figures 16, 17). Glucose levels were similar throughout each of the cultures and did not deplete as concentrated glucose feed solutions were added to each of the cultures as needed. However, there were significant differences in the lactate levels observed from day 9 until harvest. The lower pH process conditions supported higher lactate levels, with the pH 6.70 + / - 0.10 condition supporting the highest final lactate level of 6.8 g / L and the pH 6.95 + / - 0.05 condition supporting the lowest final lactate level of 2.2 g / L. These results suggest that there are significant differences in metabolism as a function of process pH control for YB2 / 0 cells and that this is the first time this relationship has been reported for this particular cell line.

[0418] Dissolved carbon dioxide levels were also monitored throughout each of the respective cell culture conditions (Figure 18). High levels of pCO2 have previously been shown to have a detrimental effect on overall process performance using other mammalian cell lines (de Zengotita, V., et al., Cytotechnology 28: 213-227 (1998)). The in-process pCO2 levels observed under bioreactor conditions are a function of both the pH setpoint, the level of lactate in the culture (higher levels of lactate support a more acidic environment), as well as the overall air and oxygen infusion rates in the bioreactor. The same maximum air infusion rate was used in each of the 10 L bioreactor cultures evaluated. The oxygen infusion rate was controlled by the bioreactor controller, and higher flow rates are typically required for cultures with higher VCD levels. The YB2 / 0 cell culture results indicate that the lowest pH control conditions supported the highest levels of pCO2 observed. That is, the pH 6.70 + / - 0.10 cultures yielded peak pCO2 levels of 146 mmHg midway through the culture, while all higher pH control conditions supported pCO2 levels < 100 mmHg. Collectively, these results emphasize that higher pH control conditions supported the lowest pCO2 levels, as well as the highest cell growth profiles and antibody titers described below.

[0419] Antibody titers were monitored through each of the above-described laboratory-scale cell culture conditions (Figure 19). Across the entire pH control range evaluated, antibody titers began to increase between days 4 - 5 of the process. Antibody titers increased similarly to approximately the same level until day 10, after which they began to diverge. Higher pH culture conditions continued to increase until the collection time point, with the measured collection day titers being 1.26 g / L at pH condition 6.85 + / - 0.05 and 1.19 g / L at pH condition 6.95 + / - 0.05 g / L. Lower pH culture conditions did not demonstrate a significant titer increase after day 10, yielding collection titers of 1.0 g / L at pH condition 6.80 + / - 0.03 and 0.79 g / L at pH condition 6.70 + / - 0.10. The overall results indicate that as the post-shift pH control range increases to higher values, pCO2 decreases and, similar to the behavior highlighted in Figure 13, the resulting antibody productivity increases.

[0420] In other cell lines, an increase in culture pH has been shown to support a decrease in productivity (Jiang, R. et al., Bioprocess Biosyst Eng 41: 1731-1741 (2018)), and a decrease in culture pH has been shown to support an increase in productivity (Seo, J.S. et al., Appl Microbiol Biotechnol. 97: 5283-5291 (2013)), so these results were surprising. In other reports using CHO cell lines, the results were mixed. In two reports, an increase in recombinant productivity was seen at higher pH values, but only in the pH range of 7.00 - 7.20 (Yoon, S.K. et al., Biotechnol. Bioeng. 89:345-356 (2005); Kim, H.S. et al., J Microbiol and Biotech. 17:712-720 (2007)). In another report, it was found that culture pH had no effect on productivity (Hennicke, J. et al., New Biotechnology 50:20-26 (2019)). In a different report using a CHO cell line expressing an antibody, pH 6.8 was found to support a 2-fold higher titer compared to pH 7 (Oguchi, S. et al., Animal Cell Technology; Basic & Applied Aspects 13:169-172 (2003)). There are no available reports describing the relationship of culture pH in YB2 / 0 cells, and this study is considered the first demonstrated example.

[0421] The expressed antibody (TG-1101) was protein A purified from each of the 10L bioreactor cultures and analyzed for product quality including N-linked glycan (protein glycosylation), charge heterogeneity (iCIEF), and size (SEC). Table 7 summarizes the N-linked glycan and highlights that the optimal pH control range (6.95+ / -0.05) supported the lowest level of % fucosylation (18.4%). Additionally, as the pH control range increased (i.e., 6.80+ / -0.03 or higher), the total amount of sialylated N-glycans increased from 0% to 1%, effectively introducing new N-glycan species into the profile. Thus, the protein fucosylation level can be nominally controlled for this specific YB2 / 0 cell line by pH-mediated pCO2 control. Table 8 summarizes the charge heterogeneity results that were not significantly affected by different pH and pCO2 conditions. Table 9 summarizes the results of the antibody size characterization and highlights that there were no changes in the individual species across different pH and pCO2 conditions.

[0422]

Table 8

[0423]

Table 9

[0424]

Table 10

[0425] Purified antibodies (TG-1101) derived from each of the 10L bioreactor cultures were also tested in various binding assays (CD20 antigen, Fc gamma receptor, complement component 1q) and Fc effector activity assays (ADCC, CDC). The results of each of these tests are shown in Table 10. The FcγRIIIa-158V, CD20 and C1q binding results were comparable across each of the culture conditions evaluated. The ADCC and CDC bioassay results were also shown to be comparable across each of the culture conditions. Collectively, these results emphasize that the higher cell growth and antibody titers induced by higher process pH control and the associated lower pCO2 control did not have a detrimental effect on the functional activity of the antibody.

[0426]

Table 11

[0427] Conclusion In this example, the inventors demonstrated an alternative way to increase process performance (cell growth, recombinant protein productivity) and a method to modulate protein glycosylation from a cell culture process using the YB2 / 0 cell line. In this example, the process parameter of cumulative pCO2 exposure in cell culture was found to have a significant role in both cell growth and antibody productivity. The culture pH was confirmed to be the primary driver for the culture pCO2 level. Both a higher pH control range (>6.80) and a lower pCO2 level control (<100 mmHg) in culture were found to support high process titers (≧1.0 g / L) for this particular generally unused mammalian expression system. In the bioreactor high productivity state, along with the control of the % fucosylation level, the demonstrated success of the binding and effector function / activity of the expressed anti-CD20 antibody was shown.

[0428] [Example 3] Commercial scale manufacturing process for producing TG-1101 in YB2 / 0 rat hybridoma cells In this example, a manufacturing process for TG-1101 (TG Therapeutics, Inc.) expressed in YB2 / 0 rat hybridoma cells at 15,000 L is described. The overview of the manufacturing process for TG-1101 is illustrated in the flow diagram of Table 12.

[0429] [Table 12]

[0430] In summary, the production of each batch of TG-1101 started with the thawing of the working cell bank (WCB) vials described further below. The culture was expanded through a series of shake flasks and seed bioreactors to meet the inoculum requirements of the 15,000 L production bioreactor operated in fed-batch mode. The bioreactor was harvested and clarified by centrifugation followed by depth filtration. The clarified harvest was purified by three chromatography steps including Protein A, cation exchange, and anion exchange, designed to purify TG-1101 and reduce process impurities such as host cell proteins and residual DNA. The purification process (as described in Example 4) contained steps to ensure virus safety, including virus inactivation (solvent / surfactant) and virus filtration steps. The final UFDF and formulation steps were used to concentrate and buffer exchange TG-1101 into the formulation buffer and at the desired product concentration. The drug substance ready for filling was formulated to obtain TG-1101 at a concentration of 25.0 mg / mL 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.

[0431] Additional descriptions of upstream and downstream process operations are provided below.

[0432] Expression Vector, Production Cell Line, and Cell Bank The host cell line used for the generation of the TG-1101-producing cell line was the rat hybridoma cell line YB2 / 0. The producing cell line, R603-12D11, was developed after transfection of the YB2 / 0 host cell line with the expression vector HK463-25 (containing the immunoglobulin heavy and light chain cDNA sequences of TG-1101). Figure 20 depicts the expression vector map of HK463-25 for the production of TG-1101 in a 15,000 L bioreactor.

[0433] Expression vector The expression vector HK463-25 contained various elements optimized for stable expression in the YB2 / 0 host cell line. The Rous sarcoma virus long terminal repeat (RSV LTR) promoter was used for the constitutive expression of both the heavy and light chain cDNAs. This promoter corresponds to the terminal repeat of the RSV genome containing enhancer elements in its 5’ region and has strong transcriptional activity in the YB2 / 0 cell line. Transcription termination and polyadenylation of both the heavy and light chain cDNAs were provided by the human growth hormone polyadenylation sequence (hGH polyA). To improve expression, a chimeric intron was introduced 5’ to the cDNA sequence of each antibody chain. This intron is optimized for splicing and is composed of a 5’ donor sequence derived from human beta-globin and a 3’ acceptor sequence derived from the Ig heavy chain variable gene. The beta-lactamase gene was provided to confer ampicillin resistance (AmpR) and enable plasmid production in Escherichia coli (E. coli). The enzyme neomycin phosphotransferase II (NeoR) was placed under the control of the SV40 promoter and confers resistance to the antibiotic G418 to the transfected cell line, thus acting as a selectable marker. Dihydrofolate reductase (Dhfr) was placed under the control of the SV40 promoter and confers resistance to methotrexate (MTX) and can similarly act as a selectable and amplifiable marker in the transfected cell line.

[0434] The HK463-25 expression vector (Figure 20) was 11.1 kb in size and contained five open reading frames for the antibody heavy chain, light chain, Dhfr, NeoR, and AmpR genes in the same orientation. The restriction sites shown in the figure were used for Southern blot analysis of the integration of the construct. The unique NotI restriction site located 3' to the NeoR gene was used for linearization of the vector prior to transfection.

[0435] Producing cell line, R603-12D11 After transfection of the host cell line, selection and screening of the transfectants, and subsequent limiting dilution cloning, the producing cell line, R603-12D11, was generated. The clones were screened, the producing cell line R603-12D11 was selected, and adapted to serum-free medium. A pre-seed stock (PSS) cell bank was prepared. A schematic of the steps involved in the generation of the producing cell line R603-12D11 is shown in Table 13.

[0436]

Table 13

[0437] A cryovial of the YB2 / 0 cell bank (YB2 / 0-301 04 / 147) was thawed and the cells were grown by dilution to a cell density of 1×10 5 cells / mL every 3 - 4 days in fresh culture medium (EMS medium with 5% FCS). Cells were seeded at a density of 2×10 5 cells / mL the day before transfection to reach the logarithmic phase prior to transfection. The expression vector HK463-25 (Figure 20) linearized with 44.5 μg of NotI was transfected into 5×10 6 cells by electroporation using Optimix reagent (Equibio) without animal components. The cells were diluted in culture medium and seeded at 100 cells / well in a 96-well plate. Selection with 1 g / L G418 in the culture medium was started 3 days after electroporation.

[0438] The transformants were first screened for titer by ELISA after selection in G418 medium. Over 3000 transformants were screened and over 200 of the best-producing wells were selected for further examination and continuous subculture. A second titer screening was performed to further reduce the number of clones. Following this screening, additional screening for antibody fucose levels was performed by ELISA. Fucose levels of less than 40% were considered desirable to provide the expected level of CD16 activation by the antibody; CD16 activation is inversely proportional to the fucose level. Further screening using a cell-based CD16 activation assay by assaying IL-2 secretion in addition to assaying the free kappa / I IgG ratio (<0.2 for ease of purification) led to the shortlisting of a total of 5 cell lines for further development.

[0439] The 5 candidate cell lines were cloned by limiting dilution at 0.4 cells / well in EMS medium with 5% FCS. The clones were screened for IgG productivity, fucose level, CD16 activation and free kappa / I IgG ratio to identify the producing cell line R603-12D11. When the selected cell line was expanded from the cloning step, it was also transferred to serum-free medium, re-screened to ensure the desired phenotype, and then a small cell bank was prepared. Next, this small cell bank was thawed and expanded to generate the R603-12D11 Pre-Seed Stock cell bank (PSS). The R603-12D11 PSS cell bank was demonstrated to be free of mycoplasma, adventitious virus and microbial contamination prior to the generation of the MCB.

[0440] Characterization of the master and working cell banks derived from the R603-12D11 producing cell line included tests for identity, genotypic and phenotypic characteristics, and the presence of adventitious agents.

[0441] Master Cell Bank (MCP) Preparation and Testing The MCB was manufactured by Henogen (later acquired by NovaSep). MCB Lot G071 / MCB / 070208 was prepared by thawing and expanding one vial of the production cell line R603 - 12D11 precryo 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). Cells were expanded for 11 days in T - flasks and roller bottles. The cell suspension was concentrated by centrifugation and aliquoted into 13 separate fractions. Each fraction was centrifuged and resuspended in freezing medium (90% EM - SF2 P500 H4 + 10% DMSO) to a target cell density of 10×10 6 cells / mL. Next, the suspended fractions were each aliquoted into 18 cryovials per fraction, resulting in a total of 234 cryovials of MCB. The cryovials were placed on dry ice and then placed into a cryobox, which was placed in a - 80°C freezer for 21 hours. The cryovials were transferred to a liquid nitrogen tank on February 19, 2007 for long - term storage and are currently stored at multiple locations. The number of cell generations from the PSS of the production cell line to the MCB is 10.4.

[0442] After the generation of the MCB lot G071 / MCB / 070208, direct inspection of the MCB and further characterization by inspection of the derived WCB were performed. Identity testing of the MCB was carried out; the test results confirmed the identity of the MCB as being of rat origin. Performance qualification was confirmed by thawing the MCB vials and monitoring cell viability. Copy number, restriction endonuclease profile, number of integration sites, integrity of the RNA coding sequence, and RNA quantification were assayed. The number of heavy and light chain copies integrated into the genome was estimated by quantitative polymerase chain reaction (Q-PCR) to be 1.13 and 2.14, respectively. Southern blot assay of plasmid integration sites demonstrated hybridization patterns comparable to those in the MCB and PSS. The number of integration sites for both the MCB and WCB was similarly measured by Southern blot to be 1 in both cell banks. In addition, chromosomal integration of the HK463-25 expression plasmid by insertion at a single locus on the middle centromeric chromosome was demonstrated by fluorescence in situ hybridization (FISH) analysis. For the MCB, the integrity of the RNA coding sequence was consistent with the reference sequence and RNA quantification was consistent with the PSS cell bank for both the heavy and light chains.

[0443] Next-generation nucleic acid sequencing (NGS) using targeted locus amplification (TLA) was performed on the MCB (Cergentis, Utrecht, Netherlands) to confirm that the expressed TG-1101 antibody has the correct amino acid sequence and to examine for low-level sequence variants. Testing of the MCB for adventitious viruses, mycoplasma, and microbial agents was performed and the acceptance criteria established for release of the MCB, including criteria for adventitious viruses, mycoplasma, and microbial agents, were met. The overall MCB test results confirmed the suitability for the establishment of the MCB.

[0444] Working Cell Bank Lot G140 / R603 / WCB001 The initial WCB was manufactured by NovaSep (after acquiring Henogen). To prepare the WCB, one vial of MCB G071 / MCB / 070208 was thawed and expanded in serum-free medium EM-SF2 P500 H4 in flasks and roller bottles over 11 days. The expanded cell suspension was concentrated by centrifugation and aliquoted into 22 identical fractions. Each fraction was centrifuged and resuspended in freezing medium (90% EM-SF2 P500 H4 + 10% DMSO) to a target cell density of 12.1×10 6 cells / mL. Next, the suspended fractions were each aliquoted into 18 cryovials, resulting in a total of 396 cryovials of WCB. The lot was named G140 / R603 / WCB001. The cryovials were placed on dry ice and then put into 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 WCB was stored at at least two different storage locations, with the manufacturer containing a smaller number of vials stored for a shorter duration. The number of cell generations from the PSS of the production cell line to the WCB was 21.4.

[0445] The lot G140 / R603 / WCB001 of WCB was tested, including identity tests. The test results confirmed the identity of the WCB as being of rat origin. Performance qualification was confirmed by thawing vials of the WCB and monitoring cell viability, doubling time, and IgG productivity. The copy number, restriction endonuclease profile, and number of integration sites were assayed. The number of heavy and light chain copies integrated into the genome was estimated by quantitative polymerase chain reaction (Q-PCR) to be 1.2 and 2.5, respectively. These results are consistent with those of the MCB. Southern blot assay of plasmid integration sites demonstrated hybridization patterns comparable in the WCB and MCB. The number of integration sites for the WCB was measured by Southern blot to be 1.

[0446] Next-generation nucleic acid sequencing (NGS) using targeted locus amplification (TLA) was performed in the WCB (Cergentis, Utrecht, Netherlands) to confirm that the expressed TG-1101 antibody has the correct amino acid sequence and to examine for low-level sequence variants. Foreign virus, mycoplasma, and microbial agent testing was performed, and all acceptance criteria established for the release of the WCB, including criteria for foreign virus, mycoplasma, and microbial agents, were met. The overall WCB test results confirmed the suitability for the establishment of the WCB.

[0447] Phenotypic characterization of the cell bank For the MCB and two WCBs generated for the manufacture of TG-1101, cell line phenotypic stability studies were performed. One vial each of the MCB, WCB lot G140 / R603 / WCB001, and new WCB lot 127646-001 was thawed and passaged over approximately 60 generations. VCD, cell viability, and titer samples were taken at each passage. At approximately 15-cell generation intervals, the cells were cryopreserved as research cell banks (RCBs). After approximately 60 generations, vials from each RCB were thawed and expanded for 7 days. On day 7, they were inoculated into shake flasks and cultured under fed-batch conditions for 12 days. The stability of each cell bank at its corresponding generation number was assessed using daily sampling of the VCD, cell viability, titer, specific productivity, and quality characteristics of the collected fed-batch cultures. Comparability in cell growth, titer, and product quality was used as a general basis for determining the phenotypic stability of the cell bank. In addition, specific productivity results exceeding 70% of the results of control cultures with fewer generations were 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 for MCB lot G071 / MCB / 070208, Table 15 for WCB lot G140 / R603 / WCB001, and Table 16 for WCB lot 127646.

[0448]

Table 14

[0449]

Table 15

[0450]

Table 16

[0451] The thawed MCB and WCB used in the production of TG-1101 at 15,000 L were examined for viable cell density and cell viability results to assess and confirm cell bank storage stability.

[0452] Upstream Process and Process Control The process flow diagrams of the upstream unit operations, including the operating control and in-process control, are provided in Table 17. Among the controls, bioburden and endotoxin were measured in the batch media of the seed and production bioreactor stages.

[0453]

Table 17

[0454] Cell Culture Medium and Feed Preparation The cell culture growth medium (CDM4Mab) and feeds (BalanCD CHO Feed4, glucose feed, and glutamine feed) were prepared using water for injection (WFI). The medium and feeds were filtered (≦0.2 μm) and placed in sterile containers and stored if necessary before use. Cholesterol lipid concentrate was supplemented to 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 addition on process days 0 and 4. The operating and in-process controls for the cell culture medium and feeds are described in Table 18.

[0455]

Table 18

[0456] Inoculum expansion The inoculum expansion step included thawing of the WCB vial and growth in shaking flasks and / or cell bags of increasing size and volume to provide sufficient cytoplasmic mass for seeding of the seed bioreactor stage. The step was carried out by growth in an inoculum expansion growth medium (CDM4Mab). To initiate the process, vials of WCB G140 / R603 / WCB001 were thawed in pre-warmed water in a 37.0 °C water bath. The thawed vial contents were transferred to pre-warmed medium and diluted to achieve a target seed density of 0.55×10 6 viable cells / mL.

[0457] The culture was placed in an initial 125 mL shaking flask and grown in a shaking incubator at 37.0 °C / 5.0% CO2 for 1 day. Every 2 - 3 days, the culture was transferred to larger volume shaking flasks and / or multiple shaking flasks for expansion. At each stage, a seeding density of 0.30×10 6 viable cells / mL was targeted. The final inoculum preparation stage consisted of a 50 L cell bag. After 2 - 3 days of growth, the viable cell density was checked and the culture was further processed to the seed bioreactor stage. The manipulations and in-process controls for inoculum expansion are described in Table 19.

[0458]

Table 19-1

[0459]

Table 19-2

[0460] Seed bioreactor The seed bioreactor stage further increases the volume and cell culture biomass prior to inoculation of the production bioreactor. The medium used in these stages was the cell density increase medium (CDM4Mab). The seed bioreactor stage was in 120L, 600L and 3000L stainless steel bioreactors. The bioreactors were equilibrated after medium addition. The dissolved oxygen and pH probes were calibrated before use. The initial bioreactor set points included temperature, pH, dissolved oxygen and agitation speed. Each bioreactor was inoculated with the cell culture from the previous stage and the culture was grown for 2 - 3 days. The seed bioreactor operation and in-process control are summarized in Table 20.

[0461]

Table 20 - 1

[0462]

Table 20 - 2

[0463] Production bioreactor The production bioreactor stage is the final cell culture process stage that further increases the volume and mass of the cell culture for the expression of the TG - 1101 antibody with acceptable product quality. The basal medium used in this stage was the growth medium (CDM4Mab) and feed addition was performed on the specified days or according to the criteria in the process. The production bioreactor was a 15,000L stainless steel bioreactor.

[0464] The production bioreactor was equilibrated after medium addition. The cell density increase medium was added up 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 speed. The bioreactor was inoculated with 0.5×10 6The cell culture from the N-1 seed bioreactor was inoculated with a viable cell density target of viable cells / mL. In the production bioreactor process, the pH was controlled by base addition and CO2 injection as required. Dissolved oxygen was controlled by oxygen and air injection as required. Antifoam was added if necessary to reduce the concern of foaming. Offline pH, pCO2, pO2, weight osmolality, and metabolites were monitored daily, as well as viable cell density (VCD) and viability. BalanCD CHO Feed4 was added on days 3, 5, 7, and 9 at a specified volume (4.0% of the initial bioreactor working volume). If the daily bioreactor sample measurement was <3.00 g / L on a specified process day, a calculated volume of glucose solution was given (up to 4.00 g / L). A fixed volume of glutamine solution was given as a bolus on day 3 (3.0% of the initial bioreactor working volume), and if the daily bioreactor sample measurement was <3.00 mM on a specified process day, the calculated volume was given (up to 4.00 mM). The production bioreactor was harvested based on either the culture duration or the cell viability criterion, whichever occurred earlier.

[0465] For the foreign substance inspection described in Example 4, an untreated bulk sample was removed prior to the clarification unit operation. The production bioreactor operation and in-process control are described in Table 21 below.

[0466]

Table 21-1

[0467]

Table 21-2

[0468] Downstream Process and Process Control A process flow diagram of downstream steps including manipulation control and in-process control is provided in Table 22. As shown in Table 22, in-process control was incorporated into the process. Among the controls, bioburden and endotoxin were measured at multiple stages of the downstream process.

[0469]

Table 22-1

[0470]

Table 22-2

[0471] Collection clarification Cell culture supernatant was collected and clarified from a 15,000 L bioreactor to remove cells and cell debris. Clarification was performed using continuous centrifugation followed by depth filtration.

[0472] The collection clarification step was operated in a room with a controlled temperature range of 17 - 25°C; the collection pool container was a jacketed tank that maintained the pool at 2 - 8°C. The centrifuge shot interval was set based on the packed cell volume percentage (PCV) and adjusted to allow 80% bowl filling. The flow rate to the centrifuge was actively controlled; the filtration feed flow was the same as the centrifuge feed flow. Process parameters included centrate backpressure, depth filtration operating pressure, inlet pressure, collection weight, bioburden, and endotoxin.

[0473] The centrate was clarified by using a three-stage filtration process that required it to pass through 0.2 μm filters (Millistak A1HC POD depth filter, 1.2 / 0.5 μm filter, and 0.45 / 0.22 μm sterile grade filter). Before use, the filters were flushed with WFI and subsequently equilibrated. The centrate was pumped through the filters, and this was monitored to ensure an acceptable backpressure. Air was used to discharge the contents in the filters, which were subsequently flushed with buffer. The clarified collection was stored at 2 - 8 °C for ≤ 11 days.

[0474] Protein A column chromatography (ProA) Protein A column chromatography was performed using MabSuRe Select resin (Cytiva) in bind / elute mode. This step provided the capture and purification of TG-1101, along with the reduction of process impurities such as cell culture components, HCP, and residual DNA, and the provision of virus safety.

[0475] The packed column was assayed for HETP performance using a sodium acetate / benzyl alcohol buffer. During production, all column manipulations were carried out at 13 - 25 °C. Before loading, the column was sanitized with 0.5 M sodium hydroxide and rinsed with WFI. The column was equilibrated with equilibration buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1). The clarified collection was mixed briefly and then loaded onto the column using a maximum of 36 g TG-1101 / L resin load, and the column was washed with 1 buffer (equilibration buffer), followed by a second wash with 2 buffers of high salt concentration (25 mM Tris, 1.2 M NaCl, 5 mM EDTA, pH 7.1), and then an additional wash using 3 buffer (equilibration buffer). The bound TG-1101 was eluted at 200 - 220 cm / h using elution buffer (25 mM sodium citrate, pH 3.6) with elution peak collection by A280 so as not to exceed 2.4 column volumes. The eluate was collected in a tank containing neutralization buffer (2.0 M Tris, pH 7.5) and filtered through a 0.2 μm filter before transferring to a different tank. The Protein A column was sanitized with 0.5 M sodium hydroxide. A maximum of 3 cycles per batch can be run; if multiple cycles are required for the Protein A process, the column was re-equilibrated with equilibration buffer for the next cycle. After sanitization, the Protein A column was neutralized with equilibration buffer and stored at 13 - 25 °C in 200 mM sodium acetate, 2% benzyl alcohol, pH 5.0. The pooled (if 2 or more cycles) and neutralized eluate was 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 or at 2 - 8 °C for ≤11 days.

[0476] Solvent / detergent virus inactivation (SDVI) Following the Protein A capture chromatography step, a solvent / detergent virus inactivation (SDVI) step was performed to inactivate potential viral agents. In the currently validated manufacturing process, the Protein A elution pool was diluted, treated with 3.5% (v / v) TnBP and 12% (w / v) polysorbate 80, mixed, and held at 24.0 - 26.0 °C for at least 120 minutes. The SDVI pool was filtered through a 0.2 μm filter, then transferred to a different tank where it was diluted with 5 mM sodium phosphate, pH 7.2 to 50 mOsm / kg and the pH was adjusted to 7.2 if necessary. After pH adjustment, the pool was held at 13 - 25 °C for ≤ 30 hours and then proceeded to the CEX column.

[0477] Cation exchange chromatography (CEX) Cation exchange column chromatography was performed using SP Sepharose Fast Flow (Cytiva) in a bind / elute mode. This step removed residual process impurities (HCP, DNA, residual polysorbate 80 and TnBP) to provide further purification of TG-1101.

[0478] The filling column was assayed for HETP performance using a sodium acetate / benzyl alcohol-containing buffer. During production, all column operations were carried out at 13 - 25°C. In the currently validated manufacturing process, prior to loading, the column was sanitized with 0.5 M sodium hydroxide and rinsed with WFI. The column was equilibrated using an equilibration buffer (20 mM sodium phosphate, pH 7.2). The virus inactivation / dilution solution was loaded onto the column at a maximum resin load of 65 g / L. The column was washed with Wash Buffer 1 (equilibration buffer), followed by a second wash with Wash Buffer 2 (equilibration buffer in the reverse direction). The bound TG-1101 was eluted using 20 mM sodium phosphate, 150 mM NaCl, pH 7.2 and collection of the elution peak by A280 monitoring. The eluate was filtered (0.2 μm) and stored at 13 - 25°C for ≤72 hours or at 2 - 8°C for ≤11 days. After elution, the column was stripped with 2 M NaCl, followed by sanitization with 0.5 M sodium hydroxide. One cycle per batch was possible. After completion, the column was sanitized (0.5 M sodium hydroxide) and stored in a storage buffer (200 mM sodium acetate, 2% benzyl alcohol, pH 5.0).

[0479] Anion exchange membrane chromatography (AEX) Anion exchange membrane chromatography was performed in flow-through mode using a Mustang Q (Pall Corporation) membrane absorber (MA) filter. This step provided further purification of TG-1101; the product flowed through the membrane and the remaining impurities (DNA, HCP and virus) were retained on the membrane. The membranes are disposable (i.e., individual membranes cannot be reused), and several membrane capsules can be used at the appropriate loading level per batch.

[0480] To perform the steps, in the currently validated manufacturing process, the eluate from the cation exchange column chromatography step was diluted with 20 mM sodium phosphate, pH 8.0, followed by adjustment to pH 8.0. The concentration was determined, and the number of cycles was calculated based on the protein concentration such that the load was 200 - 700 g TG-1101 / L membrane load. The membrane was sanitized with 0.5 M sodium hydroxide, flowed with 2 M NaCl and then WFI, and then equilibrated with equilibration buffer (20 mM sodium phosphate, 75 mM NaCl, pH 8.0) in preparation for loading. After loading, the membrane was chased with 20 mM sodium phosphate, 75 mM NaCl, pH 8.0 to maximize recovery. The collected flow-through containing the product from 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. The adjusted AEX pool was stored at 13 - 25°C for ≤72 hours or at 2 - 8°C for ≤11 days.

[0481] Virus Filtration (VF) Virus filtration was performed by filtering in series through a Viresolve pre-filter with a Viresolve Pro virus filter (Millipore Sigma) at a controlled temperature target range of 13 - 25°C. The process used sufficient filters to meet the load limit. The step was designed to remove potential viruses including small viruses such as parvovirus.

[0482] To perform the filtration, the filters were installed in series, flowed with WFI, integrity was inspected, and then sanitized with 0.5 M sodium hydroxide. Subsequently, it was flowed with equilibration buffer (25 mM sodium citrate, 154 mM NaCl, pH 6.5). The filtered Mustang Q membrane flow-through was processed through a virus reduction filter.

[0483] The protein concentration was determined and the membrane load ratio was ≤600 g / m 2It was used to confirm that it was so. After loading, the membrane was chased with the equilibration buffer, and an integrity test was performed after use. The virus filtrate was stored at 13 - 25 °C for ≤72 hours or at 2 - 8 °C for ≤11 days.

[0484] Ultrafiltration / Diafiltration (UFDF) control The UFDF step was used to concentrate the virus filtrate and buffer-exchange it into the diafiltration buffer within the operating temperature target range of 13 - 25 °C. The process used a tangential flow filter with a 30 kDa molecular weight cut-off. The device included a filter sufficient to meet a maximum loading limit of ≤250 g / m 2 To perform the operation, the membrane was disinfected with 0.5 M NaOH, rinsed with WFI, rinsed / equilibrated with the diafiltration buffer, and then the unit operation was started.

[0485] The unit operation included an initial ultrafiltration (UF1) step in which TG-1101 was first concentrated to the target of 39 mg / mL. Subsequently, diafiltration (DF) with an 8 diavolume at an upper limit of ≤9.2 diavolumes into the diafiltration buffer (25 mM sodium citrate, 154 mM NaCl, pH 6.5) was performed. The ultrafiltration system was rinsed with the diafiltration buffer to maximize recovery; the rinse was transferred to the formulation container and combined with the UF / DF pool. The membrane was 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 was stored at 15 - 25 °C for ≤18 hours.

[0486] Formulation, filtration, and filling The formulation step included the addition of concentrated polysorbate 80 in the formulation buffer to achieve the final drug substance formulation at a controlled temperature target range of 17 - 25°C. The step was carried out by adding a stabilizing buffer (25 mM sodium citrate, 154 mM NaCl, 10 g / L polysorbate 80, pH 6.5). After the addition of the stabilizing buffer, the pool was diluted to a target of 23.5 - 26.5 mg / mL at 25 mM sodium citrate, 154 mM NaCl, 700 mg / L polysorbate 80, pH 6.5, resulting in a drug substance ready for filling in a formulation buffer of 25 mM sodium citrate, 154 mM NaCl, 0.07% polysorbate 80, pH 6.5.

[0487] The formulated bulk drug substance was transferred, 0.2 μm filtered, and placed into a 6 L Celsius® FFT bag in a closed disposable system 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.

[0488] [Example 4] Detection of Viruses or Extraneous Agents in the 15,000 Commercial - Scale Production of TG - 1101 in YB2 / 0 Rat Hybridoma Cells The extraneous agent and virus safety of TG - 1101 was ensured by several strategies in a multi - faceted approach. The strategies included preventing the introduction of extraneous agents into the manufacturing process, removing them by dedicated processing steps, and inspecting for them at appropriate points in the process. Additionally, testing for adventitious viruses was performed to confirm the absence of adventitious viruses in cell line / cell bank development and manufacturing. Virus clearance studies confirmed the ability of the manufacturing process to remove viruses.

[0489] In the manufacture of TG - 1101, the comprehensive virus safety strategy consisted of the following approaches and activities:

[0490] Raw material procurement There are no raw materials of animal or biological origin that are directly used in the upstream manufacturing process of TG-1101. For example, CDM4Mab cell growth medium and BalanCD-CHO Feed 4, which are cell culture media and feeds used in production, are proprietary commercially available cell culture media. Neither medium contains animal components, and the components are not identified as having come into contact with raw materials of animal origin in their manufacture. In the downstream manufacturing process, the Protein A chromatography resin is not derived from materials of animal origin, and the cationic chromatography resin did not contain any raw materials derived from animal origin.

[0491] Inspection of the production cell line under development and the resulting cell banks (MCB, WCB, and EPCB) The cell bank system for TG-1101 was a two-tier system consisting of one master cell bank (MCB) and working cell banks (WCBs). The cell bank (i.e., MCB and WCB) adventitious agent testing included mycoplasma, microbial, and non-endogenous and adventitious virus testing. Additional adventitious agent testing (microbial and viral) was performed at the end of the production cell bank (EPCB) derived from the WCB.

[0492] Adventitious agent assessment during the manufacture of TG-1101 drug substance Specifically, routine mycoplasma, microbial, and viral adventitious agent testing of the unprocessed bulk harvest (from the production bioreactor) was performed throughout the manufacturing process, along with in-process bioburden and endotoxin testing.

[0493] Virus clearance studies Examinations on virus clearance in representative scale - down models demonstrated 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 for retroviruses. Three additional non - specific model viruses were selected to provide various virus characteristics and sizes, as well as resistance to chemical inactivation. Mouse minute virus (MVM), a model for parvovirus, was selected as a virus with a small size and high resistance to chemical inactivation, and is also a potential virus for mammalian cell line contamination. Pseudorabies virus (PRV) and reovirus type 3 (Reo 3) were also examined for virus clearance.

[0494] The TG - 1101 manufacturing process included specific, orthogonal, and dedicated virus inactivation / removal steps, including a solvent / surfactant inactivation step and a virus filtration step. The manufacturing process also included several chromatography steps that also contributed to virus inactivation / removal during the manufacturing process. Overall, the control of exogenous substances in the manufacturing process provided a high degree of assurance that the TG - 1101 drug substance had appropriate safety from the perspective of exogenous substances.

[0495] [Example 5] Effect of post - shift pH on process performance and product quality of the TG - 1101 upstream manufacturing process Background Throughout the development of the upstream portion of the commercial TG - 1101 manufacturing process ( "Process C"), numerous experiments and data analyses were performed. Culture pH was identified as an important parameter that could affect process performance and product quality. The pH control of the commercial manufacturing process run (15,000 L) ( "Process C2", commercial process) at Samsung Biologics (SBL) follows the control strategy shown in Table 23.

[0496]

Table 23

[0497] Based on both the 15 kL and 20 kL GMP manufacturing results and the scale - down model (10 L) results, the cumulative cell exposure to pH after the pH shift on day 3 (72 hours) was identified as being particularly important for process performance. This latter calculated parameter is defined as the "integrated pH2 difference" and can be conceptually viewed as the shaded section shown in Figure 21.

[0498] Materials and Methods Cell Culture The mammalian cell expression system used was the TG - 1101 YB2 / 0 rat hybridoma cell line expanded from the working cell bank lot #G140 / R603 / WCB001 or lot #127646 described above. After cryovial thawing of cells from either WCB, separate cell expansions were performed in shaking flask vessels of increasing volume and disposable cell bag bioreactors. Cells were expanded in CDM4Mab (Cytiva) cell culture medium supplemented to 1× cholesterol level using 1000× cholesterol (Thermo) over multiple passages. After further cell expansion in the seed bioreactor culture, when sufficient cell inoculation material was generated, the production culture was run in fed - batch mode. In the fed - batch production culture, BalanCD CHO Feed 4 (Fuji) was added at fixed volumes on days 3, 5, 7, and 9 of the process. In addition, an essential glutamine feed was added on day 3. Glutamine and glucose feeds were added according to pre - established criteria. Generally, the cell culture process conditions were similar across each of the different cultures analyzed, except for normal or intentionally introduced drift in the culture pH. The cell culture growth medium, feed medium, and feed solutions were prepared in a manner consistent with the spirit of GMP manufacturing.

[0499] During the duration of the production bioreactor culture, in-process monitoring was performed. Among these samples, daily measurements of viable cell density (VCD) and cell viability by an automated cell counter (ViCell XR, Beckman Coulter) were included. A Nova Flex2 (Nova Biomedical) instrument was used for metabolite measurements (glucose, lactate, glutamine, ammonia). If necessary, a Nova pHOx (Nova Biomedical) and / or a Siemens blood gas analyzer were used to measure offline pH, pO2 and pCO2. A Nova Flex 2 (Nova Biomedical) and an Advanced Instruments osmometer were used to measure weight osmolality. For titer and product quality measurements, cells were removed from the culture, clarified by centrifugation, and maintained in storage until ready to be analyzed.

[0500] Protein analytical and functional assays N-glycan N-linked glycans are cleaved from the product by enzymatic deglycosylation using PNGase F and labeled with the fluorescent compound 2-aminobenzamide (2-AB). The labeled glycans are developed using a hydrophilic interaction partition mode ultra-high performance liquid chromatography (HILIC-UPLC) column equipped with a fluorescence detector (fluorescence excitation at 360 nm and emission at 428 nm). 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 chromatograms is identified based on retention time and compared to peaks in glycan standards confirmed by mass spectrometry.

[0501] Results and conclusions The post-shift pH (pH2) follows a generally consistent trajectory from the perspective of that trend. That is, around days 7-8 of the production bioreactor culture, lactate begins to increase to a recognizable amount, whereby the culture pH drops towards the lower end of its control range. The speed and timing of this pH drop vary between process C GMP manufacturing batches and 10L scale-down model cultures (Figure 22). As a result, the cumulative cell exposure to pH over each of these cultures is somewhat different, and as a result, the integrated pH2 difference also varies. In terms of the lower the integrated pH2 difference, the higher the integrated viable cell density (IVCD), the integrated pH2 difference has an inverse relationship with the cumulative number of cells generated in the production bioreactor (Figure 23). As a result of a larger number of cells, the integrated pH2 difference also has an inverse relationship with the titer result at collection (Figure 24). As a result of a larger amount of TG-1101 constituitively secreted from the cells, the % fucosylation is generally lower in cultures demonstrating a lower integrated pH2 difference (Figure 25). These results indicate that if the goal is to reduce % fucosylation, such as when the shift is made to transfer the TG-1101 manufacturing process from process A / B to process C, this can be achieved by introducing process changes that increase TG-1101 productivity. Minimization of the integrated pH2 difference is one of the means to do so.

[0502] The post-shift pH is directly controlled in the commercial TG-1101 manufacturing process using a pH control strategy that prevents the % fucosylation from deviating from the drug substance (DS) release specification. When the post-shift pH is controlled within its control range at commercial manufacturing scale, the observed % fucosylation results are relatively intransient in the range of 22-37%.

[0503] [Example 6] The unique glycosylation signature of an anti-CD20 antibody produced in YB2 / 0 rat hybridoma cells at 15,000L commercial scale Using the manufacturing method disclosed herein (e.g., as described in Example 3, etc.), anti-CD20 antibodies were produced on a 15,000 L scale from the source antibody TG-1101. The anti-CD20 antibodies were found to contain a unique glycosylation profile. Without being bound by theory, the relative distribution of various N-glycans, or the individual sugar residues present in such N-glycans, can determine the biological and clinical properties of the anti-CD20 antibody compositions provided herein. See the co-pending (and co-owned) U.S. Patent Application No. 63 / 347,852, titled "ANTI-CD20 ANTIBODY COMPOSITIONS," filed on June 1, 2022, the U.S. Patent Application No. 63 / 421,078, filed on October 31, 2022, and the U.S. Patent Application No. 63 / 445,082, filed on February 13, 2023, the entire contents of which are incorporated herein by reference. Together with the described anti-CD20 antibody compositions, the anti-CD20 antibody compositions described herein can be used to treat cancer (e.g., hematological cancer) and autoimmune disorders (e.g., RMS).

[0504] The glycosylation profile of a sample of the anti-CD20 antibody was determined by measuring the fluorescently labeled N-glycans (the fluorescent label is 2-aminobenzamide) enzymatically cleaved from the anti-CD20 antibody protein using PNGase F. The labeled glycans were developed using a provided hydrophilic interaction column. The glycans flowed through a fluorescence detector after separation. Peak identification from the test sample chromatogram was based on retention time and identified relative to the peaks in the glycan standards 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 Figure 26.

[0505] The glycosylation profile of the anti-CD20 antibody was assayed by intact mass spectrometry (LC-MS) under non-reducing conditions. Samples of the anti-CD20 antibody were first exchanged into an MS-compatible buffer in a chromatography step using SEC and a mobile phase containing TFA, acetonitrile, and water. Next, the samples were introduced into an ESI-QTOF for intact mass analysis. The mass spectra were deconvoluted and peaks were assigned based on mass. The relative abundance of each anti-CD20 antibody provided herein containing N-glycans was calculated by obtaining the abundance of the N-glycans and dividing by the total abundance of all identified peaks. The results are provided in Table 24 below.

[0506] [Table 24-1]

[0507] [Table 24-2]

[0508] The anti-CD20 antibodies provided herein can be represented 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.

[0509] In some embodiments, 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: (a) 0.3% - 2% G0-GN; (b) 0.1% - 2% G0F-GN; (c) 0.1% - 1% G1-GN; (d) 5% - 20% G0B; (e) 5% - 30% G0F; (f) 0.1% - 1.5% Man5; (g) 1% - 15% G0FB; (h) 1% to 13% G1; (i) 0.5% to 10% G1’; (j) 0.5% to 6% G1B; (k) 0.5% to 12% G1F; (l) 0.1% to 3% G1F’; (m) 0.1% to 3% G1FB; (n) 0.1% to 2% G2; and (o) 0.1% to 2% G2F.

[0510] In a more specific embodiment, the anti-CD20 antibody provided herein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 species of N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 0.3% to 0.6% G1-GN; (d) 9.5% to 14.1% G0B; (e) 12.8% to 19.7% G0F; (f) 0.4% to 0.7% Man5; (g) 5.1% to 7.0% G0FB; (h) 5.7% to 6.4% G1; (i) 2.7% to 3.3% G1’; (j) 1.4% to 2.0% G1B; (k) 2.6% to 4.2% G1F; (l) 1.1% to 1.6% G1F’; (m) 1.1% to 1.8% G1FB; (n) 0.5% to 0.7% G2; and (o) 0.3% to 0.5% G2F.

[0511] In an even more specific embodiment, the anti-CD20 antibody provided herein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 species of N-glycans within the following relative abundance ranges: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1’; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F’; (m) 1.3 G1FB; (n) 0.5% G2; and (o) 0.3% G2F.

[0512] In some embodiments, the anti-CD20 antibodies provided herein comprise an N-glycan profile having a relative abundance of about 0.3% to about 2% G0-GN, about 0.8% to about 1.1% G0-GN, or about 0.9% G0-GN. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile having a relative abundance of about 0.1% to about 2% G0F-GN, about 0.5% to about 1.1% G0F-GN, or about 0.8% G0F-GN. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile having a relative abundance of about 0.1% to about 1% G1-GN, 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 having a relative abundance of about 5% to about 20% G0B, about 5% to about 15% G0B, about 9.5% to about 14.1% G0B, about 10.9% G0B, or about 10% G0B. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile having a relative abundance of about 5% to about 30% G0F, about 12.8% to about 19.7% G0F, or about 17.0% G0F. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile having a relative abundance of about 0.1% to about 1.5% Man5, 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 having a relative abundance of about 1% to about 15% G0FB, about 5.1% to about 7.0% G0FB, or about 6.0% G0FB. In certain embodiments, the anti-CD20 antibody comprises an N-glycan profile having a relative abundance of about 1% to about 13% G1, 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 having a relative abundance of about 0.5% to about 10% G1’, 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 having a relative abundance of about 0.5% to about 6% G1B, 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 that includes a relative abundance 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 antibody comprises an N-glycan profile that includes 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 antibody comprises an N-glycan profile that includes a relative abundance of from about 0.1% to about 3% G1FB, from about 1.1% to about 1.8% G1FB, or about 1.3 G1FB. In certain embodiments, the population of anti-CD20 antibodies comprises an N-glycan profile that includes 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 antibody comprises an N-glycan profile that includes 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.

[0513] In some aspects, the anti-CD20 antibodies provided herein comprise an N-glycan profile that includes relative abundances of from about 0.3% to about 2% G0-GN, from about 0.1% to about 2% G0F-GN, from about 0.1% to about 1% G1-GN, from about 5% to about 20% G0B, from about 5% to about 30% G0F, from about 0.1% to about 1.5% Man5, from about 1% to about 15% G0FB, 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.

[0514] In some embodiments, the anti-CD20 antibodies provided herein have an N-glycan profile comprising relative abundances of about 0.8% to about 1.1% G0-GN, about 0.5% to about 1.1% G0F-GN, about 0.3% to about 0.6% G1-GN, about 9.5% to about 14.1% G0B, about 12.8% to about 19.7% G0F, about 0.4% to about 0.7% Man5, about 5.1% to about 7.0% G0FB, about 5.7% to about 6.4% G1, about 2.7% to about 3.3% G1’, about 1.4% to about 2.0% G1B, about 2.6% to about 4.2% G1F, about 1.1% to about 1.6% G1F’, about 1.1% to about 1.8% G1FB, about 0.5% to about 0.7% G2, and about 0.3% to about 0.5% G2F. In some embodiments, Man5 is the only high-mannose N-glycan in the N-glycan profile.

[0515] In some embodiments, the anti-CD20 antibodies provided herein have an N-glycan profile comprising relative abundances of about 0.9% G0-GN, about 0.8% G0F-GN, about 0.5% G1-GN, about 10.9% G0B, about 17.0% G0F, about 0.6% Man5, about 6.0% G0FB, about 6.1% G1, about 2.9% G1’, about 1.6% G1B, about 3.2% G1F, about 1.3% G1F’, about 1.3 G1FB, 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.

[0516] In some embodiments, the anti-CD20 antibodies provided herein have an N-glycan profile comprising a relative abundance ratio of G1 to G0 N-glycans of about 0.1 to about 0.15. In some embodiments, the anti-CD20 antibodies provided herein have an N-glycan profile comprising a relative abundance ratio of G1F to G1 N-glycans of about 0.5 to about 0.9.

[0517] The invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0518] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited through this application are hereby expressly incorporated by reference in their entirety for any purpose, in the same manner as the references cited therein.

Claims

1. A method for producing an anti-CD20 monoclonal antibody in rat hybridoma cells, the method comprising the step of culturing the rat hybridoma cells in at least 10,000 L of cell culture having a culture pH of about 6.5 to about 7.55, wherein the rat hybridoma cells contain an expression vector comprising a polynucleotide encoding the anti-CD20 monoclonal antibody.

2. The method according to claim 1, wherein the culture pH is approximately 6.5 to approximately 7.0 and is optionally set to the second or third day of the cell culture.

3. The method according to claim 1, wherein the culture pH is approximately 7.0 to approximately 7.55, and optionally the culture pH of approximately 7.0 to approximately 7.55 is set to the 0th to 3rd day of the cell culture, and / or optionally the culture pH is reduced to approximately 6.5 to approximately 7.0 on the 2nd or 3rd day of the cell culture.

4. i) The rat hybridoma cells expressing the anti-CD20 monoclonal antibody are cultured in a culture medium of known composition and free of animal-derived components (ADCF), and / or ii) The method described above, ２ The further step includes controlling the level to less than approximately 300 mmHg, and / or iii) The method further comprises the steps of collecting the anti-CD20 monoclonal antibody produced by the rat hybridoma cells, purifying the anti-CD20 monoclonal antibody by affinity chromatography and / or ion exchange chromatography, wherein the affinity chromatography optionally includes protein A purification, formulation the anti-CD20 monoclonal antibody produced by the rat hybridoma cells into a pharmaceutically acceptable formulation, and / or iv) The cell culture is carried out in a commercial-scale bioreactor of 10,000 L, 15,000 L, 20,000 L or 25,000 L, and / or v) The rat hybridoma cells are YB2 / 0 rat hybridoma cells. The method according to claim 1.

5. An initial temperature setting point of approximately 37°C, further including an initial temperature setting point set from day 0 to day 1 of culture, and optionally including a second temperature setting point of approximately 35°C, set from the end of day 1 to day 3 of culture. The method according to claim 1, further comprising an optional third temperature setpoint of approximately 32°C to approximately 33°C, which is set on the third day of culture and maintained until collection.

6. The cell culture was performed under the following conditions: i) an initial temperature setting point of approximately 37°C, which is set from day 0 to day 1 of culture; a second temperature setting point of approximately 35°C, which is set from the end of day 1 to day 3 of culture; and a third temperature setting point of approximately 32.5°C, which is set on day 3 of culture and maintained until collection; ii) Culture pH between approximately 6.5 and approximately 7.55; and iii) Culture pCO2 less than approximately 300 mmHg ２ The method according to claim 1, including the method described in claim 1.

7. The aforementioned anti-CD20 monoclonal antibody a) Heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 The method according to claim 1, including the method described in claim 1.

8. The method according to claim 7, wherein the anti-CD20 monoclonal antibody comprises a heavy chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 7 and a light chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 8, and optionally 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.

9. The aforementioned anti-CD20 monoclonal antibody is as follows: i) Approximately 10-20% galactosylated glycans; and / or ii) Approximately 20-40% fucosylated glycans The method according to claim 1, comprising an N-glycan profile containing one or both of the above.

10. The method according to claim 9, wherein the N-glycan profile comprises about 10-20% galactosylated glycan and about 23-36% fucosylated glycan.

11. The method according to claim 1, wherein the anti-CD20 monoclonal antibody comprises an N-glycan profile including (i) at least about 10% bisected N-glycan and / or (ii) less than 5% sialylated glycan and / or (iii) 0.1% to 1.5% Man5 N-glycan.

12. A method for producing an anti-CD20 monoclonal antibody in the culture of rat hybridoma cells on a commercial scale, a) The steps of preparing and thawing the rat hybridoma cell bank containing the anti-CD20 monoclonal antibody, b) The cultures of the rat hybridoma cells obtained from the cell bank are divided into a series of shaking flasks (125 mL, 500 mL, 3 L, 3 × 3 L shaking flasks and a 50 L cell bag) in terms of size and volume, at least 0.30 × 10 ６ The steps include increasing the target seeding density by individual living cells / mL, c) A step of further increasing the volume and mass of the cell culture by processing the cell culture with a series of seed bioreactors (120 L, 600 L, and 3,000 L), d) The step of inoculating the cell culture from the 3,000 L seed bioreactor into a commercial-scale production bioreactor, e) The step of collecting the cell culture supernatant from the commercial-scale bioreactor, f) A step of clarifying the recovered cells by continuous centrifugation followed by deep filtration, g) A step of purifying the anti-CD20 monoclonal antibody by protein A capture column chromatography, h) A step of inactivating viral factors by a solvent surfactant virus inactivator (SDVI) and A method that includes this.

13. i) The commercial-scale production bioreactor is operated in fed-batch mode, ii) A drilled hole (10) gas spurger having a 4.0 mm orifice diameter is used in the commercial-scale production bioreactor, and / or iii) The method further comprises purification by cation exchange chromatography (CEX) and anion exchange chromatography (AEX), and / or iv) The method further comprises viral filtration (VF) for removing potential viruses, and / or v) The method further comprises ultrafiltration / dialysis filtration (UFDF), and / or vi) The method further comprises the step of preparing a bulk drug substance formulation containing the anti-CD20 monoclonal antibody, which includes adding polysorbate 80 to a formulation buffer to prepare a bulk drug substance formulation, and / or vii) The anti-CD20 monoclonal antibody is produced on a commercial scale of approximately 10,000 L to approximately 25,000 L, and / or viiii) The above method produces an antibody protein collection titer of approximately 0.5 g / L to approximately 1.5 g / L. The method according to claim 12.

14. The method according to claim 12, wherein the rat hybridoma cells are YB2 / 0 cells.

15. The aforementioned anti-CD20 monoclonal antibody a) Heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 The method according to claim 12, including the method described in claim 12.

16. The method according to claim 15, wherein the anti-CD20 monoclonal antibody comprises a heavy chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 8, and optionally the anti-CD20 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.

17. The aforementioned anti-CD20 monoclonal antibody is as follows: i) Approximately 10-20% galactosylated glycans; and / or ii) Approximately 20-40% fucosylated glycans The method according to claim 12, comprising an N-glycan profile containing one or both of the above.

18. The method according to claim 17, wherein the N-glycan profile comprises about 10-20% galactosylated glycan and about 23-36% fucosylated glycan.

19. The method according to claim 12, wherein the anti-CD20 monoclonal antibody comprises an N-glycan profile including (i) at least about 10% bisected N-glycan and / or (ii) less than 5% sialylated glycan and / or (iii) 0.1% to 1.5% Man5 N-glycan.

20. An anti-CD20 monoclonal antibody prepared according to the method described in any one of claims 1 to 19.

21. A rat hybridoma cell bank composition, a) The composition is a rat hybridoma master cell bank (MCB) composition containing YB2 / 0 cells expressing an anti-CD20 monoclonal antibody, wherein the anti-CD20 monoclonal antibody has the following parameters: i) Approximately 11 to 13 x 10 ６ Peak live cell density of cells / mL; ii) Collection titer of approximately 650 to 720 mg / L; iii) Approximately 30% to 38% fucosylation; iv) Approximately 97% to 99% monomers detected by size exclusion chromatography (SEC); v) Approximately 1.5% to 2% dimers detected by SEC; vi) Aggregates at levels undetectable to approximately 3% by SEC; vii) Fragments at levels undetectable to approximately 1% by SEC; viiii) Approximately 25% to approximately 30% acidic isoforms detected by imaging capillary isoelectric focusing (iCIEF); ix) Approximately 38% to 49% of the major isoforms detected by iCIEF; and / or x) Approximately 20% to 36% basic isoforms detected by iCIEF Having at least two of the following, or b) The composition is a rat hybridoma working cell bank (WCB) composition containing YB2 / 0 cells expressing an anti-CD20 monoclonal antibody, wherein the anti-CD20 monoclonal antibody has the following parameters: i) Approximately 11 to 28 x 10 ６ Peak live cell density is the number of cells / mL; ii) Collection titer of approximately 420 to 1280 mg / L; iii) Percent fucosylation of approximately 18% to 40%; iv) Approximately 97% to 99% monomers detected by size exclusion chromatography (SEC); v) Approximately 1% to 2% dimers detected by SEC; vi) Aggregates at levels undetectable to approximately 2% by SEC; vii) Fragments at levels undetectable to approximately 1% by SEC; viiii) Acidic isoforms of approximately 19% to 31% detected by imaging capillary isoelectric focusing (iCIEF); ix) Approximately 34% to 62% of the major isoforms detected by iCIEF; and / or x) Approximately 14% to 38% basic isoforms detected by iCIEF Having at least two of the following: composition.

22. A method for producing an anti-CD20 monoclonal antibody using the MCB or WCB composition described in claim 21.

23. The aforementioned anti-CD20 monoclonal antibody a) Heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and b) Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4; light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 A composition of MCB or WCB according to claim 21, or the method according to claim 22, comprising:

24. The MCB or WCB composition or method according to claim 23, wherein the anti-CD20 monoclonal antibody comprises a heavy chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 7; and a light chain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 8, and / or the anti-CD20 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.

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