Methods of reducing host cell proteins in recombinant protein production

Abstract

Provided herein are methods of reducing expression of host cell proteins during a mammalian cell culture process using peroxisome proliferator–activated receptor γ (PPARγ) modulator and / or an autophagy-inducing compounds.

Description

METHODS OF REDUCING HOST CELL PROTEINS IN RECOMBINANT PROTEIN PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 729,097, filed December 6, 2024, the entire contents of which are incorporated by reference herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on April 14, 2025, is named 26028WOPCT_SL.xml and is 2,842 bytes in size.FIELD OF THE INVENTION

[0003] Provided herein are methods of reducing host cell proteins (HCP) (e.g., lipases) during recombinant protein production. Also provided herein are methods of improving polysorbate (e.g., PS-80 and PS-20) stability in formulations of recombinant proteins produced from mammalian cell culture (e.g., Chinese Hamster Ovary (CHO) cells).BACKGROUND OF THE INVENTION

[0004] In bioprocessing and manufacturing of recombinant proteins (e.g., antibodies), host cell proteins (HCP) (e.g.. lipases) constitute part of the impurities that are often difficult to remove from the antibodies. Such impurities can cause various issues in the safety and efficacy of biopharmaceuticals. Regulatory agencies throughout the world require that biopharmaceutical products meet certain acceptance criteria, including the level of impurities and tests for detection and quantification of impurities.SUMMARY OF THE INVENTION

[0005] Biologies are commonly produced using Chinese hamster ovary7(CHO) cell lines under controlled processes to ensure high quality7and purity. Despite multiple purification steps, small quantities of residual host cell proteins (HCPs) often persist, posing challenges to the final drug product. While upstream processes are the primary source of these impurities, the specific biological pathways influenced by7these processes that affect HCP levels remain to be elucidated The present invention provides, among other things, methods of producing recombinant proteinswith significantly less polysorbate-degrading HCP contaminants, resulting in significantly improved stability in polysorbate containing protein formulations.

[0006] The present disclosure provides methods of reducing expression of host cell proteins during a mammalian cell culture process to improve polysorbate stability in recombinant protein production.

[0007] In one aspect, the present disclosure provides a method of reducing expression of host cell proteins during a mammalian cell culture process, comprising: (a) establishing a mammalian cell culture comprising cells engineered to express a recombinant protein; and (b) contacting the cell culture with a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound. In one aspect, the present disclosure provides a method of reducing expression of host cell proteins during a mammalian cell culture process, comprising: (a) establishing a mammalian cell culture comprising cells engineered to express a recombinant protein; and (b) contacting the cell culture with a peroxisome proliferator-activated receptor y (PPARy) modulator. In one aspect, the present disclosure provides a method of reducing expression of host cell proteins during a mammalian cell culture process, comprising: (a) establishing a mammalian cell culture comprising cells engineered to express a recombinant protein; and (b) contacting the cell culture with an autophagy -inducing compound.

[0008] In one aspect, the present disclosure provides a method of producing a recombinant protein comprising culturing a population of mammalian cells engineered to express the recombinant protein in a medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy' -inducing compound.

[0009] In one aspect, the present invention provides a method of reducing expression of host cell proteins, comprising: contacting a mammalian cell culture comprising cells engineered to express a recombinant protein with a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy' -inducing compound.

[0010] In one aspect, the present disclosure provides a method of producing a recombinant protein comprising culturing a population of mammalian cells engineered to express the recombinant protein in a medium comprising an autophagy -inducing compound.

[0011] In some embodiments, the PPARy modulator is a PPARy antagonist.

[0012] In some embodiments, the PPARy antagonist is SR1664, GW9662, or SRI 1023.

[0013] In some embodiments, the PPARy antagonist is SR1664.

[0014] In some embodiments, culturing the mammalian cells with SRI 664 decreases mannosylation levels of N-glycans on the recombinant protein. In some embodiments, culturing the cells with SRI 664 decreases mannosylation levels of N-glycans on the recombinant proteinby at least 3%, at least 4%, at least 5%, at least 6%, at least 7% at least 8%, or at least 9% relative to control mammalian cells cultured without SRI 664.

[0015] In some embodiments, culturing the cells with SRI 664 reduces the total lipase activity by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%. at least 28%, at least 29% or at least 30% relative to control mammalian cells cultured without SRI 664.

[0016] In some embodiments, culturing the mammalian cells with SRI 664 improves polysorbate stability by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% relative to control mammalian cells cultured without SRI 664.

[0017] In some embodiments, polysorbate stability is measured by the amount of intact polysorbate in a sample obtained from a culture treated with SRI 664 relative to the amount of intact polysorbate in a control sample obtained from a cultured without SR 1664.

[0018] In some embodiments, culturing the cells with SR1664 reduces the levels of one or more host cell proteins selected from the group consisting of PLA2G7, CTSO, MMP19, TGFB1 and SIAE relative to control mammalian cells cultured without SRI 664.

[0019] In some embodiments, culturing the cells with SR1664 reduces the protein levels of one or more PPARy-target genes relative to control mammalian cells cultured without SRI 664.

[0020] In some embodiments, the PPARy-target genes are Fabp4, Lrpl and Pla2g7.

[0021] In some embodiments, the PPARy-target genes are decreased by at least 20 %, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% relative to control mammalian cells cultured without SRI 664.

[0022] In some embodiments, culturing the mammalian cells with SRI 664 reduces the protein levels of LPL relative to control mammalian cells cultured without SRI 664.

[0023] In some embodiments, the PPARy modulator is an inverse agonist.

[0024] In some embodiments, the PPARy inverse agonist is SR2595, BAY-4931 or BAY-0069.

[0025] In some embodiments, the PPARy inverse agonist is SR2595.

[0026] In some embodiments, culturing the mammalian cells with SR2595 reduces the level PLA2G7 and CTSA relative to control mammalian cells cultured without SR2595.

[0027] In some embodiments, the PPARy modulator is a PPARy irreversible antagonist.

[0028] In some embodiments, the PPARy modulator is GW9662, T0070907 or SR16832.26028

[0029] In some embodiments, the PPARy modulator is a thiazolidinedione, a phenylacetic acid derivative, a fatty acid, a prostaglandin, or a component of oxidized low-density lipoprotein.

[0030] In some embodiments, the PPARy modulator is selected from aleglitazar, farglitazar, muraglitazar. or tesaglitazar, pioglitazone, rosiglitazone, rivoglitazone, and troglitazone.

[0031] In some embodiments, the PPARy modulator is ciglitazone, darglitazone, englitazone, isaglitazone (MCC-555), pioglitazone, rosiglitazone, troglitazone, tularik, BRL49653, CLX- 0921, 5-BTZD, GW-0207, LG-100641, or LY-300512.

[0032] In some embodiments, the autophagy-inducing compound is an Autophagy -Inducing Peptide (AIP).

[0033] In some embodiments, the AIP is a TAT-beclin 1 peptide.

[0034] In some embodiments, the AIP comprises the amino acid sequence RRRQRRKKRGYGGDHWIHFTANWV (SEQ ID NO: 1) or YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID NO: 2).

[0035] In some embodiments, the autophagy-inducing compound is 3-Methyladenine (3-MA).

[0036] In some embodiments, culturing the mammalian cells with 3-MA increases afucosylation levels of N-glycans on the recombinant protein.

[0037] In some embodiments, culturing the mammalian cells with 3-MA improves polysorbate stability by at least 20%. at least 21%. at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% relative to control mammalian cells cultured without 3- MA.

[0038] In some embodiments, polysorbate stability is measured by the amount of intact polysorbate in a sample obtained from a culture treated with 3-MA relative to the amount of intact polysorbate in a control sample obtained from a culture without 3-MA.

[0039] In some embodiments, the autophagy-inducing compound Torin 1, rapamycin.

[0040] In some embodiments, the host cell protein is a polysorbate-degrading enzyme (PSDE).

[0041] In some embodiments, the PSDE is PLA2G7 or SIAE.

[0042] In some embodiments, the host cell protein is PLA2G7, SIAE, CTSO, MMP19, TGFB1, or CTSA

[0043] In some embodiments, the host cell protein is CLU, CPD, CTSB, CTSD, CTSZ, CTSA, HTRA1, MMP14, MMP19, MMP9, TGFB1, PPT1. LPL. ABHD14A, PLA2G7, SIAE, PLA2G15, PREPLPL, PLA2G15, and SIAE.

[0044] In some embodiments, the host cell protein is CTSZ, CTSD, CLU, CPD, and TGFB1 .

[0045] In some embodiments, the mammalian cells are Chinese Hamster Ovai ' (CHO) cells.26028

[0046] In some embodiments, the recombinant protein is an antibody or antibody fragment thereof, a chimeric protein, or an enzyme. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is a peptide.

[0047] In one aspect, the present disclosure provides a mammalian cell culture medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator.

[0048] In one aspect, the present disclosure provides a mammalian cell culture medium comprising an autophagy -inducing compound.

[0049] In some embodiments, the autophagy-inducing compound is an AIP present at a concentration of about 1-32 pM.

[0050] In some embodiments, the autophagy-inducing compound is 3-MA present at a concentration of about 2-20 mM.

[0051] In one aspect, the present disclosure provides a method of increasing afucosylated glycans on a recombinant protein comprising culturing mammalian cells expressing the recombinant protein in the presence of 3-MA.

[0052] In one aspect, the present disclosure provides a method of decreasing mannosylation levels on a recombinant protein comprising of comprising culturing mammalian cells expressing the recombinant protein in the presence of SRI 664.

[0053] In one aspect, the present disclosure provides a method of reducing polysorbate degradation in a formulation comprising culturing a mammalian cell expressing a recombinant protein in the presence of 3-MA, AIP, or SR-1664. In one aspect, the present disclosure provides a method of reducing polysorbate degradation in a formulation comprising culturing a mammalian cell expressing a recombinant protein in the presence of 3-MA. In one aspect, the present disclosure provides a method of reducing polysorbate degradation in a formulation comprising culturing a mammalian cell expressing a recombinant protein in the presence of AIP. In one aspect, the present disclosure provides a method of reducing polysorbate degradation in a formulation comprising culturing a mammalian cell expressing a recombinant protein in the presence of SR- 1664.

[0054] In one aspect, the present disclosure provides a composition obtainable by the methods described herein, wherein the composition comprises a recombinant protein.

[0055] In one aspect, the present disclosure provides a composition obtainable by the methods described herein, wherein the composition comprises a recombinant protein.

[0056] In one aspect, the present disclosure provides methods, media, and compositions that reduce host cell proteins (HCPs), selectively lower polysorbate-degrading enzy mes (PSDEs), and improve product quality7during production of recombinant proteins — particularly monoclonalantibodies — in Chinese Hamster Ovary (CHO) cells. The approach modulates cellular pathways via peroxisome proliferator-activated receptor gamma (PPARy) modulators and autophagy -modulating compounds to attenuate problematic HCPs, decrease total lipase activity, and enhance polysorbate stability without detrimental impacts on charge variants or core glycosylation attributes.

[0057] In one aspect, the present disclosure provides methods of reducing HCP expression during mammalian cell culture include establishing a CHO cell culture expressing a recombinant protein and contacting the culture with a PPARy modulator and / or an autophagy-modulating compound under conditions effective to reduce HCPs measured in harvest or Protein A pool. In some embodiments, the recombinant protein is a monoclonal antibody. In some embodiments, the PPARy modulator is a PPARy antagonist, inverse agonist, or irreversible antagonist. In some embodiments, the autophagy -modulating compound is 3-methyladenine (3-MA) or an Autophagy -Inducing Peptide (AIP).

[0058] In one aspect, the present disclosure provides representative PPARy modulators include SRI 664 (antagonist) and SR2595 (inverse agonist). In some embodiments, SRI 664 is administered at about 1 pM on Days 0 and 2 in batch culture, or at about 1 x 10-15 mol per cell for six days in fed-batch. In some embodiments. SR2595 selectively reduces PLA2G7 and CTSA without significantly affecting IgG titer. In some embodiments. SRI 664 reduces PPARy-target proteins — including FABP4, LRP1, and PLA2G7 — by at least 20% relative to vehicle control and lowers levels of high-risk HCPs such as CTSO, MMP19, TGFB1, and SIAE. In some embodiments, gene set enrichment analysis indicates depletion of lipid catabolic proteins upon SRI 664 treatment. In some embodiments, SRI 664 reduces total lipase activity measured by a fluorescent Lipase Activity Assay (LAA) normalized per mL, per titer, and per 1VCD (approximately 27% reduction in Protein A pools), improves polysorbate stability by at least 30% (about 34% over two weeks), and trims high-mannose glycans (Man5) as measured by HILIC-UPLC glycan profiling, without significantly altering charge variant profiles by HP-IEX. In some embodiments. SRI 664 does not significantly alter cell growth, viability, or IgG titer under batch conditions.

[0059] In one aspect, the present disclosure provides representative autophagy -modulating compounds include AIP comprising SEQ ID NO: 1 (RRRQRRKKRGYGGDHWIHFTANWV) and 3-MA. In some embodiments, AIP is used at about 1-2 pM added on Day 0. In some embodiments, 3-MA is used at about 5 mM added on Day 3 (batch) or applied during fed-batch up to 12 days. In some embodiments, autophagy modulation yields proteome-wide reductions in PSDEs and high-risk HCPs confirmed by DIA LC-MS proteomics and Cygnus third-generationELISA. In some embodiments, 3-MA reduces PSDEs LPL, PPT1, PLA2G7, PLA2G15, and SIAE, and high-risk HCPs CTSZ, CTSD, CLU, CPD, and TGFB1, while decreasing LAA-measured lipase activity across normalization schemes. In some embodiments, in ambr250 fed-batch cultures, 3-MA increases specific productivity (Avg Qp) relative to control and elevates the proportion of afucosylated N-glycans on the antibody from about 4% to about 7% by HILIC-UPLC analysis.

[0060] In one aspect, the present disclosure provides mammalian cell culture media comprising a PPARy modulator or an autophagy -modulating compound. In some embodiments, the medium comprises SR1664 or SR2595 at effective dosing to reduce PLA2G7 by at least 20% in CHO monoclonal antibody production. In some embodiments, the medium comprises 3-MA at about 5 mM or AIP at about 1-2 pM to reduce PSDE levels and increase afucosylation.

[0061] In one aspect, the present disclosure provides compositions obtainable by the described methods. In some embodiments, the compositions exhibit improved polysorbate stability and reduced lipase activity in Protein A pools, and decreased PLA2G7 and SIAE levels and lower high-mannose glycan content compared to control processes lacking SRI 664.

[0062] In one aspect, the present disclosure demonstrates that targeted modulation of PPARy signaling and autophagic pathways during CHO cell culture reduces problematic HCPs, selectively lowers PSDEs, and improves downstream product quality — including polysorbate stability and favorable glycan attributes — during monoclonal antibody manufacture.

[0063] In one aspect, the present disclosure provides CHO cells engineered to express a monoclonal antibody are cultured in the presence of an autophagy-modulating compound selected from 3-MA or an AIP, thereby reducing PSDEs and high-risk HCPs in the harvest and Protein A pool. In some embodiments, 3-MA is present at about 5 mM added on Day 3 of batch or applied across fed-batch for up to 12 days. In some embodiments, the AIP comprises SEQ ID NO: 1 (RRRQRRKKRGYGGDHWIHFTANWV) at about 1-2 pM added on Day 0. In some embodiments, DIA LC-MS proteomics and Cygnus third-generation ELISA confirm reductions in PSDEs (LPL, PPTL PLA2G7, PLA2G15, SIAE) and high-nsk HCPs (CTSZ, CTSD, CLU, CPD, TGFB1). In some embodiments, lipase activity measured by a fluorescent LAA, normalized per mL, per titer, and per IVCD, is significantly reduced. In some embodiments, in ambr250 fed-batch, 3-MA increases specific productivity (Avg Qp) despite lower titer / IVCD and raises antibody afucosylation from about 4% to about 7%, as quantified by HILIC-UPLC glycan profiling.

[0064] In one aspect, the present disclosure provides PLA2G7 being selectively reduced during CHO culture by contacting the cells with a PPARy modulator selected from a PPARy antagonistor inverse agonist. In some embodiments, SRI 664 is dosed at about 1 pM on Day 0 and Day 2 in batch or at about 1 x10-15 mol per cell for six days in fed-batch. In some embodiments, SRI 664 reduces PPARy-target proteins FABP4, LRP1, and PLA2G7 by at least 20% relative to vehicle control; lowers HCPs such as CTSO, MMP19, TGFB1, and SIAE; and depletes proteins involved in lipid catabolism as indicated by gene set enrichment analysis. In some embodiments, under batch conditions, SRI 664 does not significantly alter cell growth, viability, or IgG titer.

[0065] In one aspect, the present disclosure provides producing monoclonal antibodies in the presence of SRI 664 or 3-MA yields improved polysorbate stability in the Protein A pool (PAP) or drug substance by at least 30% relative to control. In some embodiments, polysorbate stability improvement is about 34% over two weeks. In some embodiments, total lipase activity in PAP is decreased by about 27% with SRI 664. In some embodiments, charge variant profiles (HP-IEX) remain unchanged, and glycan profiles exhibit desirable changes, including SR1664-mediated trimming of high-mannose glycans (Man5) without deleterious alterations.

[0066] In one aspect, the present disclosure provides co-administration of 3-MA and SRI 664 results in additive reductions in lipase activity and HCP levels compared to either agent alone. In one aspect, media formulations comprising SRI 664 at batch-effective concentrations (about 1 pM) or fed-batch dosing equivalents (about 1 xlO’15mol per cell), and media comprising 3-MA at about 5 mM, are provided to achieve reductions in PLA2G7 and PSDEs and to increase antibody afucosylation. In some embodiments, compositions obtainable by these methods display improved polysorbate stability and reduced lipase activity' in Protein A pools, together with reduced PLA2G7 and SIAE and decreased high-mannose glycan content relative to control processes lacking SRI 664.

[0067] Collectively, in one aspect, the present disclosure establishes that PPARy antagonism and autophagy modulation during CHO monoclonal antibody production provide a practical, upstream strategy' to mitigate PSDEs and high-risk HCPs, improve dow nstream purification robustness, and enhance product quality attributes without compromising growth, viability, titer, or charge variant profiles.BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1A-1B shows proteome wide impact of selected autophagy -inducing compounds and specific productivity (qP) enhancing compounds in batch culture. FIG. 1 A shows the percentage change of select Host Cell Proteins (HCPs) in treated conditions compared to control (red indicates % increase and blue indicates % decrease). FIG IB shows volcano plots of differentially expressed genes with problematic HCPs highlighted (P < 0.05).

[0069] FIG. 2A-2C shows process output from a 250 mL bioreactor run (in ambr®250). The effect of various treatments on titer (FIG. 2A), integral viable cell density (IVCD) (FIG. 2B), and specific productivity (Avg Qp) (FIG. 2C) were quantified.

[0070] FIG. 3A-3C shows lipase activity of harvested cell culture fluid (HCCF) samples of the bioreactor run described in FIG. 2A-2C. The effect of various treatments on lipase activity normalized to sample volume (FIG. 3 A), titer mU / mg (FIG. 3B), and per cell (mU / mL / VCD) (FIG. 3C) were quantified.

[0071] FIG. 4A-4B shows lipase activity from Protein A purified samples. The effect of various treatments on lipase activity normalized to sample volume (FIG. 4A) and titer mU / mg (FIG. 4B) were quantified.

[0072] FIG. 5A-5B shows HCP levels from Protein A purified samples. The effect of various treatments on HCP normalized to sample volume (FIG. 5A) and titer (FIG. 5B) were quantified.

[0073] FIG. 6A-6B shows mAb quality attributes upon treatment with PPARy antagonist (SR- 1664) or Autophagy inducer (3 -MA) or a combination of SR- 1664 and 3 -MA or adenosine supplementation (qP enhancer). The effect of various treatments on the glycosylation profile (FIG. 6A) and charge variant profiles(FIG. 6B) of Protein A purified samples are shown.

[0074] FIG. 7A-7G show s selective reduction of PSDE levels in HCCF of batch culture with PPARy antagonist (SR- 1664). SRI 664 did not significantly affect cell growth (FIG. 7A), cell viability (FIG. 7B), or IgG titer (FIG. 7C). FIG. 7D shows protein levels of select PPARy-target genes. Student’s t-test. ns: not significant; *: p < 0.05. FIG. 7E shows comparison of HCP levels between vehicle-control and SR1664-treatment groups, showing selective reduction of PLA2G7 and SIAE. The horizontal line indicates adjusted p value < 0.05. Gene set enrichment analysis (GSEA) of differentially abundant proteins between vehicle-control and SR1664-treatment groups, show s significant depletion of cellular lipid catabolic process (FIG. 7F) and fold changes of its associated proteins (FIG. 7G). Error bars represent the standard error of mean. nVehicle = 6. nSR1664 = 3.

[0075] FIG. 8A-8C shows selective reduction of PSDE levels in batch culture HCCF with a PPARy inverse agonist. SR2595 did not significantly affect IgG titer (FIG. 8A). FIG. 8B shows protein levels of select PPARy-target genes. Student’s t-test. ns: not significant; *: p < 0.05. FIG. 8C shows comparison of HCP levels between vehicle-control and SR2595 -treatment groups, showing selective reduction of PLA2G7 and CTSA. The horizontal line indicates adjusted p value < 0.05. Error bars represent the standard error of mean. nVehicle = 6. nSR2595 = 3.26028

[0076] FIG. 9 shows intact PS-80 levels in PAP after two-week incubation expressed as a percentage of its level before incubation. Error bars represent the standard error of mean. Student’s t-test. ns: not significant; *: p < 0.05; **: p < 0.01. nVehicle = 4. nSR1664 = 3.DETAILED DESCRIPTION OF THE INVENTION

[0077] In the biologies manufacturing process, polysorbates may be used as an excipient in the final drug substance formulations to improve the stability of proteins during manufacture, shipment, and storage. Polysorbates (which are (atty acid esters of polyoxyethylene sorbitan) can improve bioproduct stability by reducing aggregation and particle formation, specifically due to interfacial stresses, and surface adhesion of the active ingredient. However, polysorbates, can undergo chemical or enzyme mediated degradation to release long-chain fatty acids. Enzyme mediated degradation can occur by ester hydrolysis due to the presence of small quantities of uncleared host cell lipases or esterases in the final drug substance. Polysorbate degradation can decrease the effectiveness of the surfactant in protecting the active pharmaceutical ingredient (API) and lead to turbidity and particle formation in the formulation over time, rendering a product noncompliant, limiting its shelf life. Polysorbate degradation products may pose potential risks to patient safety. By decreasing or eliminating HCPs (e.g., cellular lipases / esterases) responsible for the enzymatic degradation of polysorbate detergents, the shelf life of recombinantly produced bioproduct formulations containing polysorbate detergents can be improved. Increased shelf life is important to ensure supply of biologic products reducing waste and enabling efficient distribution networks.

[0078] International Patent Application Publications WO 2017 / 053482. WO 2016 / 138467. WO 2018 / 039499, and WO 2015 / 095568 describe methods of reducing the expression of problematic HCPs in mammalian cells, including various lipases / esterases, each of which are hereby incorporated by reference in their entirety. US20220267369A1 describes methods of separating host cell lipases from a production protein in chromatographic processes, the entirety of which is hereby incorporated by reference.

[0079] Cell line engineering approaches and downstream purification strategies to remove or reduce HCPs may not always ensure sufficient clearance of HCPs in a given process. Accordingly, the present invention is based, at least in part, on the discovery that using certain compounds (e.g., PPARy modulators and autophagy -inducing compounds) during the cell culture process, can reduce the expression of polysorbate-degrading enzymes ultimately leading to improved stability of recombinant protein formulations that include polysorbate.26028

[0080] In one aspect, the present invention provides a method of reducing expression of a host cell protein during a mammalian cell culture process, comprising: (a) establishing a mammalian cell culture comprising cells engineered to express a recombinant protein; and (b) contacting the cell culture with a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound.

[0081] In one aspect, the present invention provides a method of producing a recombinant protein comprising culturing a population of mammalian cells engineered to express the recombinant protein in a medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound.

[0082] In another aspect, the present invention provides a mammalian cell culture medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator.

[0083] In another aspect, the present invention provides mammalian cell culture medium comprising an autophagy -inducing compound.Definitions

[0084] Certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure relates. In case of conflict, the present specification, including definitions, will control.

[0085] The term “about”, when modifying the quantity (e.g., mM, or M) of a substance or composition, the percentage (v / v or w / v) of a formulation component, the pH of a solution / formulation, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and / or use of the substance or composition; through instrumental error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In this specification, “about” means a variation of ± 5% of the value.

[0086] The phrase “maintained at > 80, 85, 90, 95, or 99% of the concentration when formulated” when used in the context of measuring PS80 or PS20 stability after a period of time takes into consideration assay variability of ± 10% in measurement of the PS80 or PS20 concentration.

[0087] As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological or binding activity. Thus, it is used in the broadest sense and specifically26028 covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

[0088] In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

[0089] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bi- or multi-functional or bi- or multi-specific antibodies, the two binding sites are, in general, the same.

[0090] Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C- terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3. CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat. et al.. (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al.. (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0091] As used herein, unless otherwise indicated, “antibody fragment” or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e., antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain26028 antibody molecules, e.g., scFv; nanobodies and multispecific antibodies formed from antibody fragments.

[0092] “Chimeric antibody” refers to an antibody in which a portion of the heavy’ and / or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0093] “Human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may' contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

[0094] “Humanized antibody” refers to forms of antibodies that contain sequences from nonhuman (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially’ all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), ty pically that of a human immunoglobulin. The prefix “hum”, “hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

[0095] Embodiments of the present disclosure also include antibody fragments including, but not limited to Fc fragments, or antigen-binding fragments that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab', F(ab’)2, Fv fragments. scFv antibody fragments, scFab. disulfide- linked Fvs (sdFv), a Fd fragment.

[0096] “Comprising” or variations such as “comprise”, “comprises” or “comprised of’ are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the26028 stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the context requires otherwise due to express language or necessary' implication.

[0097] ‘‘Conservatively modified variants'’ or “conservative substitution’" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity / hydrophilicity7, backbone conformation and rigidity', etc ), such that the changes can frequently be made without altering the biological activity7or other desired property of the protein, such as antigen affinity7and / or specificity. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity.

[0098] “Consists essentially of,” and variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any7recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition.

[0099] “Framework region” or “FR” as used herein means the immunoglobulin variable regions excluding the CDR regions.

[0100] “Kabat” as used herein means an immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health. Bethesda, Md.).

[0101] “Monoclonal antibody” or “mAb” or “Mab”, as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by7Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g.,26028U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0102] The terms “mammalian cells” and “host cells” are used interchangeably herein and refer to mammalian cells which are commonly used in the production of biologies using recombinant DNA technology. For example, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293), and mouse myeloma cells, including NSO and Sp2 / 0 cells, are commonly used mammalian cells for protein expression. In some embodiments, the mammalian cell is CHO. including, but not limited to, CHO-K1, CHO pro-3, DUKX-X11, DG44, CHO-K1 GS KO, CHOK1SV or CHOK1SV GS-KO. The cell line may be also modified by the insertion, knockout or knock-down of genes that affect the critical quality attributes or other post-translational modifications of a recombinant polypeptide, or the expression of the gene encoding the recombinant protein.

[0103] In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the host cell is a CHO-K1 cell, a CHOK1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knock-out cell (glutamine synthetase), a CHOK1SV FUT8 knock-out cell, a CHOZN. or a CHO-derived cell. The CHO GS knock-out cell (e.g, GSKO cell) is, for example, a CHO-K1SV GS knockout cell (Lonza Biologies, Inc.). In some embodiments, the CHO GS knock-out cell (e.g., GSKO cell) is, for example, a CHO-K1 GS knockout cell (Horizon Discovery). The CHO FUT8 knockout cell is, for example, the Potelligent® CHOK1SV FUT8 knock-out (Lonza Biologies, Inc.). In embodiments, the host cell is a HeLa, MDCK, Sf9, Sf21. Tn5. HT1080, NB324K. FLYRD18, HEK293, HEK293T, HT1080, H9, HepG2. MCF7. Jurkat, NIH3T3, PC 12, PER.C6, BHK (baby hamster kidney), VERO, SP2 / 0, NSO, YB2 / 0, YO, EB66, C127, L cell, COS (e g, COS1 and COST), QC1-3, CHOK1, CHOK1SV, Potelligent™ (CHOK1SV FUT8-KO). CHO GS knockout, Xceed™ (CHOK1SV GS-KO). CHOS, CHO DG44, CHO DXB11, or CHOZN cell, or any cells derived therefrom.

[0104] In some embodiments, the mammalian cell is CHO-K1. In some embodiments, the mammalian cell is a CHO GS knock-out cell (e.g, GSKO cell)

[0105] The term “polysorbate” refers to nonionic surfactants which are fatty' acid esters of polyethoxylated sorbitan. Examples of polysorbates used in formulations include, but are not limited to, Polysorbate 80 (PS80), Polysorbate 20 (PS20), Polysorbate 40 (PS40), Polysorbate 60 (PS60), Polysorbate 65 (PS65), or a combination thereof. The concentration of polysorbate in the pharmaceutical compositions of the present invention, may be at about 0.01% to about 1%, about260280.01% to about 0.10%, about 0.01% to about 0.05%, about 0.02% to about 0.05% by weight in the composition.

[0106] The terms “polysorbate-degrading enzyme” and “polysorbate-degradative enzyme" (PSDE) refer to host-cell derived enzymes that can degrade polysorbate in pharmaceutical formulations.

[0107] As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0108] As used herein, the terms “at least one” item or “one or more” item each include a single item selected from the list as well as mixtures of two or more items selected from the list.

[0109] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0110] Unless expressly stated to the contrary, all ranges cited herein are inclusive; z.e., the range includes the values for the upper and lower limits of the range as well as all values in between. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. All ranges also are intended to include all included sub-ranges, although not necessarily explicitly set forth. For example, a range of pH 4.0-5.0 is intended to include pH 4.0, 4.1, 4.13, 4.2, 4.1-4.6, 4.3-4.4, and 5.0. In addition, the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination.

[0111] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in the claims.

[0112] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.26028Host Cells

[0113] The host cell can be any cell used for expressing an exogenous protein. Common host cells used in manufacturing of biopharmaceuticals include but are not limited to CHO cell, baby hamster kidney (BHK21) cell, murine myeloma NSO cell, murine myeloma Sp2 / 0 cell, human embryonic kidney 293 (HEK293) cell, fibrosarcoma HT- 1080 cell, PER.C6 cell. HKB-11 cell, CAP cell, HuH-7 cell, murine C 127 cell, and a naturally generated or genetically modified variant thereof.

[0114] In certain embodiments, the host cell is a CHO cell.

[0115] In some embodiments, the host cell is baby hamster kidney (BHK21) cell. In other embodiments, the host cell is murine myeloma NSO cell. In yet other embodiments, the host cell is murine myeloma Sp2 / 0 cell. In still other embodiments, the host cell is human embryonic kidney 293 (HEK293) cell. In certain embodiments, the host cell is fibrosarcoma HT- 1080 cell. In some embodiments, the host cell is PER.C6 cell. In other embodiments, the host cell is HKB- 11 cell. In yet other embodiments, the host cell is CAP cell. In still other embodiments, the host cell is HuH-7 cell. In certain embodiments, the host cell is murine Cl 27 cell. In some embodiments, the host cell is a naturally generated variant of the above host cell. In other embodiments, the host cell is a genetically modified variant of the above host cell.

[0116] In some embodiments, the host cell is a Vero cell.

[0117] In some embodiments, the host cell is a yeast cell.Host Cell Proteins (HCP)

[0118] The various methods provided herein apply to a broad variety of HCP. The HCP can be any endogenous protein derived from a host cell (e.g., CHO cell) during bioprocessing of a recombinant protein (e.g., an antibody or antigen binding fragment expressed in the host cell). Non-limiting examples of HCP include structural protein, functional protein, secreted protein, enzyme, such as lipase, proteinase, and kinase, etc. In some embodiments, the HCP is a structural protein. In certain embodiments, the HCP is a functional protein. In other embodiments, the HCP is a secreted protein. In yet another embodiment, the HCP is an enzyme. In one embodiment, the HCP is a lipase. In another embodiment, the HCP is a proteinase. In yet another embodiment, the HCP is a kinase. In one embodiment, the HCP is Clusterin.

[0119] Residual host cell proteins (HCPs) with hydrolytic activity, such as lipases and other esterases, including carboxylesterases, in the final drug product (DP) can lead to polysorbate degradation. The role of lipases in the degradation of polysorbates in antibody formulations has further been emphasized by Chiu et al., wherein harvested cell culture fluid (HCCF) from26028 lipoprotein lipase (LPL) knockout CHO cells reduced the PS20 and PS80 degradation as compared to wild type CHO cells (Chui et al., 2017, Biotechnol. Bioeng. 114, 1006- 1015). Also group XV lysosomal phospholipase A2 isomer XI (LPLA2), putative phospholipase B-like- 2 (PLBL2), liver carboxylesterase, phospholipase A2 group VII (PLA2G7), lysophospholipase 2 (LYPLA2), lysosomal acid lipase (LIPA), sialic acid acetylesterase (SIAE), palmitoyl-protein thioesterase 1 (PPT1) and lipoprotein lipase (LPL) were detected in activity-based protein profiling assays and final drug products indicating these enzymes as potentially critical towards PS degradation (Hall et al., 2016, Journal of Pharmaceutical Sciences, 105(5): 1633-1642; Dixit et al.. 2016, Journal of Pharmaceutical Sciences, 105(5): 1657-1666; Zhang et al., 2020, Journal of Pharmaceutical Sciences, 109(11): 3300-3307; Chiu et al., 2017, Biotechnology and bioengineering 114(5): 1006- 1015; Li et al., 2020, Anal. Chem, 93(23): 8161-8169; Graf et al., 2021, Journal of Pharmaceutical Sciences, 110:3558-3567).

[0120] In some embodiments of various methods provided herein, the lipase is a Chinese Hamster Ovary (CHO) cell lipase. In certain embodiments, the lipase is selected from the group consisting of phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), lysosomal phospholipase A2 (LPLA2), phospholipase A2 VII (LP-PLA2), and lysosomal acid lipase A (LAL). In one embodiment, the lipase is PLBL2. In another embodiment, the lipase is LPL. In yet another embodiment, the lipase is LPLA2. In one embodiment, the lipase is LP-PLA2. In another embodiment, the lipase is LAL.

[0121] In some embodiments, the host cell protein is a polysorbate-degrading enzyme (PSDE). In some embodiments, the PSDE is PLA2G7. In some embodiments, the PSDE is SIAE.

[0122] In some embodiments, the host cell protein is PLA2G7, SIAE, CTSO. MMP19. TGFB1, or CTSA. In some embodiments, the host cell protein is CTSO. In some embodiments, the host cell protein is MMP19. In some embodiments, the host cell protein is TGFB1. In some embodiments, the host cell protein is CTSA.

[0123] In some embodiments, the host cell protein is CLU, CPD, CTSB, CTSD, CTSZ, CTSA, HTRA1, MMP14, MMP19, MMP9. TGFB1, PPT1. LPL. ABHD14A, PLA2G7, SIAE, PLA2G15, and PREPLPL.

[0124] In some embodiments, host cell protein is CLU. In some embodiments, host cell protein is CPD. In some embodiments, host cell protein is CTSB. In some embodiments, host cell protein is CTSD. In some embodiments, host cell protein is CTSZ. In some embodiments, host cell protein is CTSA. In some embodiments, host cell protein is HTRA1. In some embodiments, host cell protein is MMP14. In some embodiments, host cell protein is MMP19. In some embodiments, host cell protein is MMP9. In some embodiments, host cell protein is TGFB1. In26028 some embodiments, host cell protein is PPT1. In some embodiments, host cell protein is LPL. In some embodiments, host cell protein is ABHD14A. In some embodiments, host cell protein is PLA2G7. In some embodiments, host cell protein is SIAE. In some embodiments, host cell protein is PLA2G15. In some embodiments, host cell protein is PREPLPL.

[0125] In some embodiments, the host cell protein is CTSZ, CTSD. CLU, CPD, and TGFB1. In some embodiments, host cell protein is CTSZ. In some embodiments, host cell protein is CTSD / In some embodiments, host cell protein is CLU. In some embodiments, host cell protein is CPD. In some embodiments, host cell protein is TGFB1.Peroxisome proliferator-activated receptor y (PPARy) Modulators

[0126] The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. PPARy is involved in regulating the differentiation of adipocytes, where it is highly expressed. It also has a key role in systemic lipid homeostasis. PPARy is modulated by a range of synthetic and naturally occurring substances including thiazolidinediones (TZDs), phenylacetic acid derivatives, fatty acids, prostaglandins and components of oxidized low-density lipoproteins.

[0127] The present invention is based at least in part on the use of PPARy modulator for reducing host cell proteins in a mammalian cell culture.

[0128] In some embodiments, the PPARy modulator is a PPARy antagonist.

[0129] In some embodiments, the PPARy antagonist is SRI 664. In some embodiments, SRI 664 is present in the cell culture at a concentration of about 1 pM to about 7.5 pM. In some embodiments, SRI 664 is present in the cell culture at a concentration of about 1.0 pM to about1.5 pM, about 1.5 pM to about 2.0 pM, about 2.0 pM to 2.5 pM, about 2.5 pM to about 3.0 pM, about 3.0 pM to about 3.5 pM, about 3.5 pM to about 4.0 pM, about 4.0 pM to about 4.5 pM, about 4.5 pM to about 5.0 pM, about 5.0 pM to about 5.5 pM, about 5.5 pM to about 6.0 pM, about 6.0 pM to about 6.5 pM, about 6.5 pM to about 7.0 pM, or about 7.0 pM to about 7.5 pM.In some embodiments. SR1664 is present in the cell culture at a concentration of about 1.0 pM, about 1.5 pM, about 2.0 pM, about 2.5 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about4.5 pM, about 5.0 pM, about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, or about 7.5 pM.

[0130] In some embodiments, the PPARy antagonist is SR 11023.

[0131] In some embodiments, the PPARy modulator is an inverse agonist.

[0132] In some embodiments, the PPARy inverse agonist is SR2595.26028

[0133] In some embodiments, SR2595 is present in the cell culture at a concentration of about 1 pM to about 7.5 pM. In some embodiments, SR2595 is present in the cell culture at a concentration of about 1.0 pM to about 1.5 pM, about 1.5 pM to about 2.0 pM, about 2.0 pM to2.5 pM, about 2.5 pM to about 3.0 pM, about 3.0 pM to about 3.5 pM, about 3.5 pM to about4.0 pM, about 4.0 pM to about 4.5 pM, about 4.5 pM to about 5.0 pM. about 5.0 pM to about5.5 pM, about 5.5 pM to about 6.0 pM, about 6.0 pM to about 6.5 pM, about 6.5 pM to about7.0 pM, or about 7.0 pM to about 7.5 pM. In some embodiments, SR2595 is present in the cell culture at a concentration of about 1.0 pM, about 1.5 pM, about 2.0 pM, about 2.5 pM, about 3.0 pM, about 3.5 pM. about 4.0 pM, about 4.5 pM, about 5.0 pM. about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, or about 7.5 pM.

[0134] In some embodiments, the PPARy inverse agonist is BAY-4931. In some embodiments, the PPARy inverse agonist is BAY-0069.

[0135] In some embodiments, the PPARy modulator is an irreversible antagonist. In some embodiments, the PPARy modulator is GW9662. In some embodiments, the PPARy modulator is T0070907. In some embodiments, the PPARy modulator is SRI 6832.

[0136] In some embodiments, the PPARy modulator is a thiazolidinedione. In some embodiments, the PPARy modulator is a phenylacetic acid derivative. In some embodiments, the PPARy modulator is a fatty acid. In some embodiments, the PPARy modulator is a prostaglandin. In some embodiments, the PPARy modulator is a component of oxidized low-density lipoprotein.

[0137] In some embodiments, the PPARy modulator is selected from aleglitazar, farglitazar, muraglitazar, or tesaglitazar. In some embodiments, the PPARy modulator is selected from pioglitazone, rosiglitazone, rivoglitazone, and troglitazone.

[0138] In some embodiments, the PPARy antagonist is a glitazone. In some embodiments, the PPARy antagonist is ciglitazone, darglitazone, englitazone, isaglitazone (MCC-555), pioglitazone, rosiglitazone, troglitazone, tularik, BRL49653, CLX-0921, 5-BTZD, GW-0207, LG-100641, or LY-300512.Autophagy-inducing compounds

[0139] Autophagy is a critical catabolic process which is necessary for maintaining cellular homeostasis. Its dysregulation has been implicated in various infectious and metabolic diseases. The present invention is based, in part, on the discovery that introducing an autophagy -inducing compound during mammalian cell culture reduces the expression of certain host cell proteins (e.g., polysorbate-degrading enzymes).26028

[0140] In some embodiments, the autophagy-inducing compound is an Autophagy-Inducing Peptide (AIP). In some embodiments, the AIP is a Tat-beclin 1 fusion peptide. In some embodiments, the AIP comprises the amino acid sequence RRRQRRKKRGYGGDHWIHFTANWV (SEQ ID NO: 1). In some embodiments, the AIP comprises the amino acid sequence YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID NO: 2).

[0141] Exemplary AIP molecules are described in US Patent No. 8,802,633, International Patent Application No. WO2022038638A1, and Shoji-Kawata et al., Nature, 2013; PMID 23364696), each of which is hereby incorporated by reference in their entirety.

[0142] In some embodiments, the AIP is present in the mammalian cell culture at a concentration of about IpM to about 50 pM. In some embodiments, the AIP is present in the mammalian cell culture at a concentration of about IpM to about 50 pM, about IpM to about 50 pM.

[0143] In some embodiments, the AIP is present in the mammalian cell culture at a concentration of about 1-50 pM, about 1-5 pM, about 6-10 pM, about 11-15 pM, about 16-20 pM, about 21-25 pM, about 26-30 pM, about 31-35 pM, about 36-40 pM, about 41-45 pM, or about 46-50 pM. In some embodiments, the AIP is present at a concentration of about 1-32 pM.

[0144] In some embodiments, the AIP is present in the mammalian cell culture at a concentration of about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 31 pM, about 32 pM, about 33 pM. about 34 pM, about 35 pM, about 36 pM, about 37 pM, about 38 pM, about 39 pM, about 40 pM, about 41 pM, about 42 pM, about 43 pM, about 44 pM, about 45 pM, about 46 pM, about 47 pM, about 48 pM, about 49 pM, about 50 pM.

[0145] In some embodiments, the autophagy-inducing compound is a small molecule. In some embodiments, the autophagy -inducing compound is Torinl. In some embodiments, Torinl is present in the cell culture medium at a concentration of about 0.25 pM to 2.00 pM, about 0.25 pM to 0.50 pM, about 0.50 pM to 0.75 pM, about 0.75 pM to 1.00 pM, about 1.00 pM to 1.25 pM, about 1.25 pM to 1.50 pM, about 1.50 pM to 1.75 pM, or about 1.75 pM to 2.00 pM. In some embodiments, Torinl is present in the cell culture medium at a concentration of about 0.25 pM, about 0.35 pM, about 0.45 pM, about 0.55 pM, about 0.65 pM, about 0.75 pM, about 0.85 pM, about 0.95 pM, about 1.05 pM, about 1.15 pM, about 1.25 pM, about 1.35 pM, about 1.4526028 pM, about 1.55 pM, about 1.65 pM, about 1.75 pM, about 1.85 pM, about 1.95 pM, about 2.00 pM.

[0146] In some embodiments, the autophagy-inducing compound is rapamycin. In some embodiments, rapamycin is present in the cell culture medium at a concentration of about 0.25 pM to 2.00 pM, about 0.5 pM to 2.00 pM, about 0.25 pM to 0.50 pM, about 0.50 pM to 0.75 pM, about 0.75 pM to 1.00 pM, about 1.00 pM to 1.25 pM, about 1.25 pM to 1.50 pM, about 1.50 pM to 1.75 pM, or about 1.75 pM to 2.00 pM. In some embodiments, rapamycin is present in the cell culture medium at a concentration of about 0.25 pM, about 0.35 pM, about 0.45 pM, about 0.5 pM to 2.00 pM, about 0.55 pM. about 0.65 pM, about 0.75 pM, about 0.85 pM. about 0.95 pM, about 1.05 pM, about 1.15 pM, about 1.25 pM, about 1.35 pM, about 1.45 pM, about 1.55 pM, about 1.65 pM, about 1.75 pM, about 1.85 pM, about 1.95 pM, about 2.00 pM.

[0147] In some embodiments, the autophagy-inducing compound is 3-Methyladenine (3-MA). In some embodiments, 3-MA is present in the cell culture medium at a concentration of about 2- 20 mM. In some embodiments, 3-MA is present in the cell culture medium at a concentration of about 2-10 mM, about 5-10 mM, about 5-15 mM, about 5-20 mM. In some embodiments, 3-MA is present in the cell culture medium at a concentration of about 2-4 mM, about 4-6 mM, about 6- 8 mM, about 8-10 mM, about 10-12 mM, about 12-14 mM, about 14-16 mM, about 16-18 mM, or about 18-20 mM. In some embodiments, 3-MA is present in the cell culture medium at a concentration of about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.EXAMPLES

[0148] The examples in this section are offered by way of illustration, and not by way of limitation.Example 1: Autophagy modulators for reducing HCP levels during cell culture process

[0149] Biologies are commonly produced using Chinese hamster ovary (CHO) cell lines under controlled processes to ensure high quality and purity. Despite multiple purification steps, small quantities of residual host cell proteins (HCPs) often persist, posing challenges to the final drug product. While upstream processes are the primary source of these impurities, the specific biological pathways influenced by these processes that affect HCP levels remain to be elucidated.

[0150] This Example demonstrates transcriptomic and proteomic comparisons between two different monoclonal antibody (mAb) expressing clones that exhibit variations in HCP levels and26028 lipase activity. This multi-omics approach identified the CLEAR (Coordinated Lysosomal Expression and Regulation) signaling pathway as a key differentiator between the clones. The CLEAR network, regulated by the master transcription factor EB (TFEB), governs lysosomal biogenesis, function, and autophagy induction. Autophagy could help contain and degrade HCPs, thereby preventing their release from the cell. Chemical modulators of autophagy, such as 3- methyladenine (3-MA) were evaluated for the ability to reduce HCP levels.

[0151] The autophagy-inducing compounds AIP (Autophagy-Inducing Peptide RRRQRRKKRGYGGDHWIHFTANWV (SEQ ID NO: 1) at I pM and 2 pM) and 3-MA (3- Methyladenine, 5 mM) were added to the culture on Day 0 and Day 3, respectively. The cultures were harvested after 7 days. The filtered cell culture fluids were subjected to DIA-based LC-MS proteomics analysis. Proteome-wide impact of selected autophagy-inducing compounds were evaluated in batch culture. As shown in Fig. 1 A, the percentage change of problematic Host Cell Proteins (HCPs) in treated conditions compared to control was determined. Volcano plots of differentially expressed genes with problematic HCPs highlighted (P < 0.05) are shown in Fig. IB.

[0152] AIP and 3-MA showed similar patterns in the proteomics profiles. A significant difference was observed with 3-MA due to its optimal concentration, whereas higher concentrations of AIP are required to see similar changes. The application of 3-MA resulted in a significant downregulation of several polysorbate-degrading enzymes (PSDEs) such as LPL, PLA2G15, and SIAE. As seen in Fig. IB, 3-MA significantly reduced the levels of several HCPs of concern, including CTSZ, CTSD, CLU, CPD, and TGFB1.

[0153] A clone having relatively high amounts of problematic HCPs was grown in various treatment conditions as shown in the Fig. 2A-2C for 12 days in ambr250 bioreactor in a fed batch mode. The effects of various treatments on Titer, Integral Viable cell density (VCD), and Specific productivity (Avg Qp) were quantified from this bioreactor run.

[0154] 3-MA, 3-MA + sr!664, and adenosine decreased the Titer as well as IVCD (Integrated viable cell density) with the 3-MA + srl664 having a strong effect. All treatment conditions increased specific productivity (Avg Qp) with 3-MA having a strong effect. Although the cell titer decreased with 3-MA containing conditions, specific productivity of the target protein increased relative to the control with 3-MA treatment.

[0155] Lipase activity from the ambr®250 bioreactor run was also evaluated to determine the effect of various treatments on lipase activity of cell culture harvest normalized to various process parameters were quantified. A Lipase Activity Assay (LAA) assay where a fluorescent probe is used to measure the lipolytic enzy me activity level was employed to measure the lipase26028 activity in the cell culture fluid. When normalized either to the volume (Fig. 3A), titer (Fig. 3B), or cell (Fig. 3C), the lipase activity was significantly reduced in the 3-MA as well as 3-MA + sr!664 condition. Similar results were observed in Protein A-purified samples subjected to the LAA assay (Fig. 4A-4B). ELISA-based third-generation Cygnus HCP detection was used to measure the levels of problematic and immunologically challenging HCPs in the Protein A- purified samples. Consistent with the results of the LAA assay, a significant reduction in HCP levels was observed under the 3-MA treatment as well as the combined 3-MA + srl664 condition (Figs. 5A and 5B).

[0156] N-Glycan analysis performed using Hydrophilic Interaction Liquid Chromatography (HILIC) on each protein A purified sample. Using N-Glycanase enzyme, the N-linked glycans were hydrolyzed from the protein. Free glycans were labeled with the dye InstantPC (iPC), and the labeled glycans were analyzed using UPLC with fluorescence detection. Charge variants w ere detected using High-Performance Ion Exchange Chromatography (HP-IEX). Treatment during cell culture with SRI 664 demonstrated a reduction in mannose levels, which is a desirable change in quality attributes. As shown in Figs. 6A-6B, treatment with the small molecules tested did not result in significant changes to the other glycosylation patterns and charge variants of each sample.

[0157] In summary, autophagy induction (e.g., using 3-MA) significantly reduces HCP levels, lipase activity, and polysorbate 80 (PS-80) degradation.Example 2: Selective reduction of polysorbate-degrading enzymes (PDSE) level with Peroxisome proliferator-activated receptor y (PPARy) antagonists

[0158] This example demonstrates selective reduction of PLA2G7 levels with PPARy antagonists during CHO cell culture. A CHO cell line engineered to express a monoclonal antibody (Clone A) w as grow n in batch mode in the absence and the presence of the non- covalent PPARy antagonist SRI 664 (1 pM at days zero and two). SRI 664 directly binds PPARy in cells and inhibits the transcriptional function. SR1664 did not significantly affect cell growth, cell viability, IgG titer, or the total lipase activity of Clone A (Fig. 7A-7C). Treatment with SRI 664 led to significant decreases by > 20 % in the protein levels of the PPARy-target genes Fabp4, Lrpl and, importantly, Pla2g7 as w ell as a decreasing trend in the LPL protein level in the harvest (Fig. 7D).

[0159] To evaluate potential proteome-wide impact of PPARy antagonism, a DIA-based quantitative proteomics approach was used to quantified 2717 HCPs (Fig. 7E). Overall, 41 protein groups were significantly more abundant in the control group whereas 14 protein groups26028 were significantly more abundant in the SRI 664 group. Besides PLA2G7, the three high-risk HCPs CTSO, MMP19 and TGFB1 and the PSDE SIAE were significantly less abundant in the SRI 664 group. Moreover, a GSEA indicated significant depletion of proteins involved in lipid catabolism (Fig. 7F, 7G), suggesting that PPARy is an important regulator of these processes in CHO cells. No significant increases in other lipase or high-risk HCP levels were observed in the SRI 664 group.

[0160] Treatment with an analogous PPARy inverse agonist (SR2595) had similar effect on PLA2G7 (Fig. 8A-8C). SR2595 did not significantly affect IgG titer (Fig. 8A) or protein levels of select PPARy-target genes (Fig. 8B). Comparison of HCP levels between vehicle-control and SR2595-treatment groups, showed selective reduction of PLA2G7 and CTSA (Fig. 8C).Example 3: Improvement of PS stability and product quality

[0161] This example demonstrates PPARy antagonist (SR 1664) and autophagy-inducing compounds (3-MA) improve PS degradation. Clone A was cultured in a fed-batch bioreactor in the absence or presence of SR1664 (1 x ICT15mol / cell) for six days or 3-MA for 12 days. SR1664 did not significantly affect either cell viability or IgG titer but reduced total lipase activity7in the harvest by approximately half (data not show n). In addition, the SRI 664 treatment group produced denser cultures and consumed more glutamine than the vehicle control group. To evaluate the impact of the lipase downregulation on downstream purification, protein A affinity chromatography was performed and the protein A pool (PAP) samples were analyzed with respect to lipase activity and product quality. Purification yield ranged 90-95 % and were comparable between the two groups. Total lipase activity was 27 % lower in the SR1664 treatment group. Polysorbate stability was improved by 34% in SRI 664 treated samples over two weeks (Fig. 9). SR1664 did not significantly alter the charge variant profile or negatively impact the glycosylation profile. Surprisingly, SRI 664 treatment caused significant Man5 trimming which is a desirable aspect in the mAb product quality. Similarly, 3-MA resulted in significant improvement of polysorbate stability relative to the control samples. In addition, its application brings significant improvement in the afucosylation level (4 % in control to ~ 7 % in 3-MA).

[0162] These results demonstrate that SR1664 and 3-MA can reduce the PS-degrading potential of PDSEs and improve desirable product quality attributes from host cells.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing expression of host cell proteins during a mammalian cell culture process, comprising:(a) establishing a mammalian cell culture comprising cells engineered to express a recombinant protein; and(b) contacting the mammalian cell culture with a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound.

2. A method of producing a recombinant protein comprising culturing a population of mammalian cells engineered to express the recombinant protein in a medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound.

3. A method of reducing expression of host cell proteins, comprising: contacting a mammalian cell culture comprising cells engineered to express a recombinant protein with a peroxisome proliferator-activated receptor y (PPARy) modulator and / or an autophagy -inducing compound.

4. The method of any one of the preceding claims, wherein the PPARy modulator is a PPARy antagonist.

5. The method of claim 4, wherein the PPARy antagonist is SR1664, GW9662, or SRI 1023.

6. The method of claim 4, wherein the PPARy antagonist is SRI 664.

7. The method of claim 6, wherein culturing the mammalian cells with SR1664 decreases mannosylation levels of N-glycans on the recombinant protein.

8. The method of claim 6, wherein culturing the mammalian cells with SRI 664 reduces the total lipase activity by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least25%, at least 26%, at least 27%, at least 28%, at least 29% or at least 30% relative to control mammalian cells cultured without SRI 664.

9. The method of any one of claims 6 or 7. wherein culturing the mammalian cell with SRI 664 improves polysorbate stability by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% relative to control mammalian cells cultured without SRI 664.

10. The method of any one of claims 6-9, wherein culturing the mammalian cells withSRI 664 reduces the levels of one or more host cell proteins selected from the group consisting of PLA2G7, CTSO, MMP19, TGFB1 and SIAE relative to control mammalian cells cultured without SRI 664.

11. The method of any one of claims 6-10, w herein culturing the mammalian cells with SRI 664 reduces the protein levels of one or more PPARy-target genes relative to control mammalian cells cultured without SRI 664.

12. The method of claim 11, wherein the PPARy-target genes are Fabp4, Lrpl and Pla2g7.

13. The method of claim 11 or 12, wherein the PPARy-target genes are decreased by at least 20 %, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34% or at least 35% relative to control mammalian cells cultured without SR1664.

14. The method of any one of claims 6-13, wherein culturing the mammalian cells with SRI 664 reduces the protein levels of low-density lipoprotein (LPL) relative to control mammalian cells cultured without SRI 664.

15. The method of any one of claims 1-3, wherein the PPARy modulator is an inverse agonist.

16. The method of claim 15, wherein the PPARy inverse agonist is SR2595, BAY-4931 or BAY-0069.

17. The method of claim 16, wherein the PPARy inverse agonist is SR2595.

18. The method of claim 17, wherein culturing the mammalian cells with SR2595 reduces the level PLA2G7 and CTSA relative to control mammalian cells cultured without SR2595.

19. The method of any one of claims 1-3, wherein the PPARy modulator is a PPARy irreversible antagonist.

20. The method of claim 19, wherein the PPARy modulator is GW9662, T0070907 or SR16832.

21. The method of any one of claims 1-3, wherein the PPARy modulator is a thiazolidinedione, a phenylacetic acid derivative, a fatty acid, a prostaglandin, or a component of oxidized low-density lipoprotein.

22. The method of any one of claims 1-3, wherein the PPARy modulator is selected from aleglitazar, farglitazar, muraglitazar, or tesaglitazar, pioglitazone, rosiglitazone, rivoglitazone, and troglitazone.

23. The method of any one of claims 1-3, wherein the PPARy modulator is ciglitazone, darglitazone, englitazone, isaglitazone (MCC-555), pioglitazone, rosiglitazone, troglitazone, tularik, BRL49653, CLX-0921, 5-BTZD, GW-0207, LG-100641, or LY-300512.

24. The method of any one of the preceding claims, wherein the autophagy -inducing compound is an Autophagy -Inducing Peptide (A1P).

25. The method of claim 24, wherein the AIP is a TAT-beclin 1 peptide.

26. The method of claim 24. wherein the AIP comprises the amino acid sequence RRRQRRKKRGYGGDHWIHFTANWV (SEQ ID NO: 1) or YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID NO: 2).

27. The method of any one of claims 1-23, wherein the autophagy -inducing compound is 3- Methyladenine (3-MA).2602828. The method of claim 27, wherein culturing the mammalian cell with 3-MA increases afucosylation levels of N-glycans on the recombinant protein.

29. The method of any one of claims 1-23, wherein the autophagy-inducing compound is Torin 1, rapamycin.

30. The method of any one of the preceding claims, wherein the host cell protein is a polysorbate-degrading enzyme (PSDE).

31. The method of claim 30, wherein the PSDE is PLA2G7 or SIAE.

32. The method of any one of claims 1-30, wherein the host cell protein is PLA2G7, SIAE, CTSO, MMP19, TGFB1, or CTSA.

33. The method of any one of claims 1-30, wherein the host cell protein is CLU, CPD, CTSB, CTSD, CTSZ, CTSA, HTRA1, MMP14, MMP19, MMP9, TGFB1, PPT1, LPL, ABHD14A, PLA2G7, SIAE, PLA2G15, PREPLPL, PLA2G15, and SIAE.

34. The method of any one of claims 1-30, wherein the host cell protein is CTSZ, CTSD, CLU, CPD, and TGFB1.

35. The method of any one of the preceding claims, wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

36. The method of any one of the preceding claims, wherein the recombinant protein is an antibody or antibody fragment thereof, a chimeric protein, or an enzyme.

37. A mammalian cell culture medium comprising a peroxisome proliferator-activated receptor y (PPARy) modulator.

38. A mammalian cell culture medium comprising an autophagy-inducing compound.

39. The mammalian cell culture of claim 38, wherein the autophagy-inducing compound is an AIP present at a concentration of about 1-32 pM.2602840. The mammalian cell culture of claim 38, wherein the autophagy-inducing compound is 3- MA present at a concentration of about 2-20 mM.

41. A method of increasing afucosylated glycans on a recombinant protein comprising culturing mammalian cells expressing the recombinant protein in the presence of 3 -MA.

42. A method of decreasing mannosylation levels on a recombinant protein comprising culturing mammalian cells expressing the recombinant protein in the presence of SRI 664.

43. A method of reducing polysorbate degradation in a formulation comprising culturing mammalian cells expressing a recombinant protein in the presence of 3-MA, AIP, or SR- 1664.

44. A composition obtainable by the method of any one of claims 2, 4-36, and 41-43, wherein the composition comprises a recombinant protein.

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