Perfusion-adapted chinese hamster ovary cells and methods of their preparation

Abstract

The present disclosure relates to a perfusion-evolved Chinese hamster ovary cell line and a method of generating said cell line.

Description

ATFHOST- 100-PCT01 -NPENGINEERED CHINESE HAMSTER OVARY CELLS AND METHODS OF THEIR USEBACKGROUND OF THE DISCLOSURERelated Applications

[0001] This application claims the priority benefit of United States Provisional Application No. 63 / 719,346, filed November 12, 2024, which is incorporated herein by reference in its entirety.Field of the Disclosure

[0002] The current disclosure generally relates to development of cell lines useful for production of biological compounds.Background

[0003] Recombinant clones generated from Chinese Hamster Ovary (CHO) host cells encounter several stressors when cultured in high intensity perfusion (HIP) processes, including susceptibility to shear stress, high cell density, (Clincke MF et al.,

[2013] Biotechnol. Prog. 29(3), pp. 754-767.; Kim JY et al.,

[2012] Appl. Microbiol. Biotechnol. 93(3) pp. 917-930, high oxygen demand, Ozturk, S.S.,

[1996] , Cytotech. 22, pp. 3-16) and increased osmolality resulting in decreased specific productivity and low long-term viability.

[0004] Exploiting the ability of CHO cells to adapt to different culture conditions, numerous directed evolution approaches involving the use of physical or chemical stressors have proven successful in selecting cells with desirable properties. Bort et al. have described a method in which CHO cells were sequentially cultured in a medium containing reduced levels of glutamine, which resulted in evolved cells with the characteristic of growing in glutamine-free medium suitable for bioprocessing. (Bort J. et al.,

[2010] Biotechnol J. 5(10), pp. 1090-97.) Prentice and co-workers selected DG44 host cells for their ability to survive in fed-batch bioreactor conditions, leading to increased peak cell density and the ability to grow in the absence of growth factors. (Prentice H.L. et al.,

[2007] Biotechnol Prog. 23(2), pp. 458-64.) Improved subcloning performance and enhanced growth behavior was reported using single cell sorting as the evolutionary pressure. (Weinguny M. et. al.,

[2020] Comput. Struct. Biotechnol. J.18, pp. 1320-29.) Adaptation of an IgG-producing CHO cell line to endoplasmic reticulum (ER) stress by continuous culturing under increasing concentration of tunicamycin resulted in higherATFHOST- 100-PCT01 -NPproductivity due to increased ER capacity. (Chandrawanshi V. et al.,

[2020] J BiotechnoL 311, pp. 49-58.) Mistry et al. described evolution of CHO host cells in the presence of hydrogen peroxide to generate oxidative stress-resistant host cells that showed improved expression of difficult-to-express proteins. (Mistry R.K. et al.,

[2021] Biotechnol Bioeng. 6.) Hatton et al. generated a novel CHO host that exhibits improved capability of recombinant polypeptide expression in suspension culture. (US Pat. No. 9,068,198).

[0005] Although various adaptation strategies have been employed to generate new CHO cells lines with improved protein production phenotypes (see, e.g., US Pat. No. 9,068,198), the development of a high producing, robust and stable clonally-derived cell lines better suited for the many challenges faced during high intensity perfusion HIP, has remained a need in the art.BRIEF SUMMARY OF THE DISCLOSURE

[0006] In some aspects, the present disclosure provides a perfusion-evolved Chinese Hamster Ovary (CHO) CAT-S cell having increased specific cellular productivity relative to an unevolved CAT-S cell, wherein perfusion-evolving the CHO CAT-S cell comprises subjecting the cell to culturing in an alternating tangential flow (ATF) filtration or tangential flow filtration (TFF) perfusion bioreactor under high cell density and glucose depleted conditions. In some aspects, the cell is cultured in an ATF perfusion bioreactor. In some aspects the cell is cultured in a TFF perfusion bioreactor. In some aspects, the perfusion-evolving comprises allowing the cell to grow at a high cell density reaching at least 1.5 x 108, 1.6 x 108, 1.7 x 108, or 1.8 x 108cells / mL. In some aspects, the perfusion-evolving comprises perfusing the CHO CAT-S cells at a rate to limit the amount of nutrients available to the cells such that all nutrients added were consumed during perfusion. In some aspects, perfusion-evolving comprises perfusing the CHO CAT-S cells at a rate to limit the amount of glucose available to the cells such that all glucose added was consumed during perfusion. In some aspects, the specific cellular productivity in the perfusion-evolved cell is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, or 1.5-fold compared to an unevolved CAT-S cell. In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of one or more proteins that regulate the kinetics of cell growth, cytoskeletal organization, and / or cellular metabolism. In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of glutathione-S-transferase. In some aspects, the cell exhibits decreased expression levels of at least one of ZNF131, NDFIP2, SORBS2, NCAM1, GCH4, WIPH, ROR1, TMEM160 IRS2, RPS18, FN1, RPL4, GPRC5A, P4HA2,ATFHOST- 100-PCT01 -NPZNF608, PRKRIP1, Ighvl6-1 or PSMC3 relative to expression levels in an unevolved CAT-S cell. In some aspects, the cell exhibits increased expression levels of at least one of RAB6B, CCDC117, DTL, VHL, HY0U1, DBN1, NFIB, MAP1LC3A, KLHL42, GAK, RLF, SPN, TM0D2, PGGT1B, UBE4B, ATAD5, TOE1, SET, RPL31, N0A1, NNMT, ZBTB38, TOE1, RPL10, UBAC2, ATCB, RPL23, UBE20, FAM102B, BRD7, SIPA1L2, MICAL2, AKNAD1, RBFA, PANK3, EVI5, ROGDI, RNFT1, BYSL, or TMEM175 relative to expression levels in an unevolved CAT-S cell. In some aspects, the cell produces lower levels of lactate compared to an unevolved CAT-S cell.

[0007] In some aspects, the present disclosure provides a method for generating perfusion-evolved Chinese Hamster Ovary (CHO) CAT-S cells, comprising:seeding an unevolved CAT-S cells in an alternating tangential flow (ATF) filtration perfusion bioreactor in a glucose-containing cell culture medium;perfusing the CAT-S cells at a rate to limit the amount of glucose available to the cells such that all glucose added was consumed between days 9 and 14 of the culture;bleeding the cells to maintain a cellular concentration of 5 x 107 cells / mL;culturing the CAT-S cells for a total of 30 days wherein the cell density exceeded 1.30 x 108 cells / mL and the glucose levels in the bioreactor were depleted; andharvesting the perfusion-evolved cells. In some aspects, the method further comprises expanding the harvested perfusion-evolved cells by culturing in a shaker flask. In some aspects, the ATF-perfusion bioreactor volume is between about 0.5-10L. In some aspects, the ATF perfusion bioreactor volume is between about 50-200L. In some aspects, the ATF perfusion bioreactor volume is between about 500-5000L. In some aspects, the evolved cells are harvested on day 30. In some aspects, the perfusion-evolved cells were maintained post-cell bleeding at a viable cell density (VCD) of 7 x 107cells / mL and 90% viability. In some aspects, the present disclosure provides a perfusion-evolved CHO CAT-S cell produced by the methods described herein. In some aspects, the perfusion-evolved CHO CAT-S cell line is deposited with American Type Culture Collection (ATCC) under deposit number PTA-127740.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0008] Figure 1A is a schematic showing the process to evolve CHO host cells in perfusion culture under high cell density and a nutrient depleted environment.ATFHOST- 100-PCT01 -NP

[0009] Figure IB shows the viable cell density (VCD) of the CHO host cells during the ATF evolution process.

[0010] Figure 1C shows cell viability of the CHO host cells during the ATF evolution process.

[0011] Figure ID shows the glucose levels during perfusion, demonstrating that the perfusion rate was maintained to limit the amount of glucose available to the cells.

[0012] Figure 2 shows the VCD and percent viability for antibody AZE1 (Fig. 2A and 2E), AZE2 (Fig. 2B and 2F), AZE3 (Fig. 2C and 2G) and AZDI (Fig. 2D and 2H), respectively in both parental and ATF evolved CHO cells.

[0013] Figure 3A-3D show that the ATF host-derived pools demonstrated a 1.5-fold increase in titer for antibody AZE1, a 1.3-fold increase in titer for AZE2, a 3-fold increase in titer for AZE3and a 2.5-fold increase in final titer for AZDI, respectively as compared to CHO pools.

[0014] Figures 3E-H shows that lactate production was found to be lower in the ATF pool cultures than the corresponding CHO pools for cells expressing AZE1, AZE2, AZE3, and AZDI, respectively.

[0015] Figure 4 shows the improved outgrowth (Fig. 4A and 4B), average titer (Fig. 4C and 4D), specific productivity (Qp) (Fig. 4E and 4F), end of run viability (EoR) (Fig. 4G and 4H), and EoR lactate production (Fig. 41 and 4J), for ATF evolved or CHO pools expressing AZE1 or AZE3, respectively.

[0016] Figures 5A and 5B show consistent intracellular antibody expression irrespective of PDL number (population doubling levels) for the two highest producing clones of AZE1 and AZE3.

[0017] Figures 5C and 5D show consistent secreted antibody productivity irrespective of PDL number (population doubling levels) for the two highest producing clones of AZE1 and AZE3.

[0018] Figures 6A and 6B show that perfusion host-derived clones El-Al and E3-A1 have increased titer compared to the parent CHO clones El-Cl and E3-C1.

[0019] Figures 6C and 6E show a higher VCD observed for El-Al than the parental CHO clones, contributing to greater titer but similar cell productivity.

[0020] Figures 6D and 6F show a similar VCD observed for E3-A1 than the parental CHO clones, but higher cell productivity, contributing to greater titer.ATFHOST- 100-PCT01 -NP

[0021] Figures 6G and 6H show that viability during the culture period remained similar between the clones expressing either of the molecules.

[0022] Figures 61 and 6J show that both El-Al and E3-A1 demonstrate lower levels of highly mannosylated product than the parent CHO clones.

[0023] Figures 7A and 7B show that 286 proteins were found to be differentially expressed in ATF evolved cells (-log2 different >0.75) at 0.05 FDR cutoff level.

[0024] Figure 8 shows the network of differentially expressed proteins in ATF evolved cells whose functions are correlated with cell morphology, cellular assembly and organization, and cellular development.

[0025] Figure 9 shows the network of differentially expressed proteins in ATF evolved cells whose functions are correlated with carbohydrate metabolism, lipid metabolism, and small molecule biochemistry.

[0026] Figure 10 shows the network of differentially expressed proteins in ATF evolved cells whose functions are correlated with cell-to-cell signaling and interaction, inflammatory response, and organismal injury and abnormalities.

[0027] Figure 11 shows the investigation of intracellular heavy chain (HC) and light chain (LC) protein expression in the pools to assess whether differences in the percentage distribution of expressing populations could be the cause of the difference in productivity observed between the two hosts. For AZE1 and AZE2, both host derived pools exhibit similar expression of HC and LC, with 89% HC+LC+ for the CHO and 92% for the ATF host and >99% for CHO and ATF pools of AZE3. For AZDI, the analysis revealed similar expression of HC and LC in both hosts, with 89% HC+LC+ for the CHO and 86% for the ATF host.DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] The maturation of cell culture, cell engineering and cell adaptation technologies over the past decade has enabled the consistent production of high titer biotherapeutics with desired critical product quality attributes using the biomanufacturing fed-batch processes.Despite the high performances derived from fed-batch, in recent years there has been a shift in process development away from fed-batch toward continuous perfusion biomanufacturing motivated by cost reductions, process intensification and improved control over product quality and process consistency.ATFHOST- 100-PCT01 -NP

[0029] Cell line development technology has undergone rapid expansions in improved efficiency and utilities. The maturation of cell culture, cell engineering, and cell adaption technologies over the past decade has enabled the consistent production of high titer biotherapuetics with desired critical product quality attributes. Those seeking to utilize cell lines for biotherapuetic production must choose between three available modes: batch, fed-batch, or perfusion biomanufacturing. Although new advancements in clone selection have largely enhanced the cell growth, viability, product titer, and cell specific productivity in fed-batch production process, inevitable byproduct accumulation causes product quality variation with time and complicates the downstream purification process. Thus, perfusion biomanufacturing is a superior choice for bioprocessing due to the cost reduction, process intensification, improved control over product quality, and process consistency.

[0030] The perfusion cultivation mode with constant renewal of the culture medium provides a stable environment to cells and the product and thereby enables significant process intensification to help meet the demand for therapeutic protein production. Inventors’ simple perfusion based directed evolution selection strategy is both an inventive use of the mechanics of the perfusion cultivation mode and development of a novel alternating tangential flow (ATF) host cell line better suited for cell line development and intensified continuous upstream processes.Definitions

[0031] As used in this specification and the enumerated paragraphs herein, the singular forms “a,” “an,” and “the” include plural reference unless the context dictates otherwise.

[0032] As used herein, the terms “substantially, “about,” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), for example, within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value of range of values.

[0033] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0034] As described herein, any concentration range percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and,ATFHOST- 100-PCT01 -NPwhen appropriate, fractions thereof (such as one tenth and one hundredth of an integer) unless otherwise indicated.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which it is disclosed is related.

[0036] As used herein, “CHO cell(s)” is an abbreviation for Chinese Hamster Ovary cells, which is an epithetical cell line derived from the ovary of the Chinese hamster (Cricetulus griseus). They are considered an ideal system for expressing recombinant proteins as the posttranslation protein modification is similar to that of human cells. Thus, they are the most widely used mammalian cells for transfection, expression, and large-scale recombinant protein production. CHO cells have a well-characterized approval history for production of clinical grade materials such as recombinant antibody therapeutics.

[0037] As used herein, “CHO-S cell(s)” is a known, commercially available (Invitrogen) suspension-adapted CHO cell line. CHO-S is used for expression of recombinant polypeptides in suspension culture and it is considered useful for protein production.

[0038] As used herein, “CAT-S cell line” is a cell line derived from a vial of CHO-K1 by adaption to growth in suspension in ACF, chemically defined (CD) medium (CD CHO, Thermo Fisher Scientific).

[0039] As used herein, “perfusion” is a process that uses a method to keep cells in a bioreactor while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed.

[0040] As used herein, “VVD” is the number of operating vessels volume per day, and is the unit of measure for the exchange of medium during cellular perfusion. By way of example, if 2L of medium is being perfused daily into a system with a 2L working volume, this would be expressed as 1 VVD.

[0041] As used herein, “perfusion-evolved” comprises subjecting the cell to culturing in an alternating tangential flow (ATF) filtration perfusion bioreactor under high cell volume and nutrient-depleted conditions. In some aspects, the cell can be subjected to culturing in a tangential flow filtration (TFF) bioreactor under high cell volume and nutrient-depleted conditions.

[0042] As used herein, “alternating tangential flow” refers to a type of cell retention system in which a filtering compartment is connected to a storage vessel at one end and aATFHOST- 100-PCT01 -NPdiaphragm at the other. The pump first moves medium from the vessel through the filter element to the pump and then reverses to send the medium from the pump through the filter and back to the vessel, creating a bi-directional or alternating flow. As used herein, “tangential flow filtration” (TFF) refers to a type of filtration method in which a solute-containing solution passes tangentially across an ultrafiltration membrane and lower molecular weight solutes are passed through a filter surface.

[0043] As used herein, “Specific Cell Productivity” (Qp) is defined as (pg protein) / cell / day. A cell line that maintains favorable productivity is vital for biopharmaceutical manufacturing processes.

[0044] As used herein, “Cell Bleeding” is the process wherein cell biomass is removed from the reactor to maintain the desired cell health and culture environment, typically described as a flow rate or percent bleed, and is the percent of VVD medium flow rate that is directed toward bleed instead of spent medium. The bleed starting point is generally determined by a sudden change in growth rate as viable cell density (VCD) increases.

[0045] As used herein, “cell viability” refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0046] As used herein, “Viable Cell Density” is the number of live cells per unit volume. In some aspects, the cells were allowed to grow to a high cell density, reaching 1.86*108cells / mL on post-inoculation day 10 with a cell viability of 90%.

[0047] As used herein, “high cell density” is defined to be about 800-1800* 105cells / mL. Generally, the maximal growth of suspension cells is mainly limited by the total amount of nutrients in the growth medium and the accumulation of growth-inhibiting compounds.

[0048] As used herein, “culture” or “cell culture” refer to a cell population, either surface-attached or in suspension that is maintained or grown in a medium under conditions suitable to survival and / or growth of the cell population. Large scale approaches, such as bioreactors, are also encompassed by “cell culture.” As used herein, “culturing” refers to growing one or more cells in vitro under defined or controlled conditions, including temperature, gas mixture, time, and medium formulation, to cells growing in suspension, for example, in flasks.

[0049] As used herein, “media,” “medium,” “cell culture medium,” “culture medium,” “growth medium,” and the like refer to a solution containing nutrients which can be used toATFHOST- 100-PCT01 -NPnourish growing cultured host cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for growth and / or survival. The solution is formulated to a pH and salt concentration optimal for cell survival and proliferation.

[0050] As used herein, “expanding” refers to culturing one or more cells in vitro for the purpose of obtaining a larger number of cells in the culture.

[0051] As used herein, “bioreactor” shall refer to a vessel for culturing cells. Any bioreactor suitable for culturing cells under perfusion culture conditions may be employed in the methods of the disclosure.

[0052] As used herein, “perfusion bioreactor” refers to a tank in which cells are cultured by continuously feeding the cells with fresh media and removing spent media while keeping cells in culture. In perfusion there are different ways to keep the cells in culture while removing spent media. For example, one way is to keep the cells in the bioreactor by using capillary fibers or membranes which the cells bind to; using filtration systems that keep the cells in the bioreactor while allowing the media to be removed; or through centrifuging.

[0053] As used herein, “shake flask” shall refer to a vessel used as a culture flask in which the medium and cell culture is constantly agitated during intubation.

[0054] The generic term “antibody” refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region and a heavy chain constant region. “Antibody” comprises five distinct classes of antibody that can be distinguished biochemically (IgG, IgM, IgA, IgE, and IgD). All five classes of antibodies are clearly within the scope of the current disclosure.

[0055] As used herein, “cell retention” refers to the goal of a perfusion culture: removing nutrient-depleted and waste product-containing media from the culture while minimizing damage to the cells. In some aspects, the type of cell retention system used is an alternating tangential flow (ATF) system. In some aspects, the type of cell retention system used is a tangential flow filtration (TFF) system.

[0056] As used herein, “lactate” refers to a salt or ester of lactic acid. High levels of lactate have clear negative impact on cell culture processes, and therefore efforts have been made to reduce lactate accumulation and / or to induce lactate consumption in the later stage of cultures.ATFHOST- 100-PCT01 -NP

[0057] As used herein, “animal-derived component” refers to natural media consisting of natural biological substances, such as plasma, serum, and embryo extract. In some aspects, no animal-derived components, including components manufactured using animal derived materials, were used in the derivation of the CAT-S ATF host from the CAT-S cell line or in thecry opreservation of the CAT-S ATF host pre-MHCB and MHCB.

[0058] As used herein, “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The terms “polypeptide” or “protein” or “product” or “product protein” are used interchangeably. As used herein, “protein” is intended to encompass a molecule comprised of one or more polypeptides, which can in some instances be associated by bonds other than amide bonds. On the other hand, a protein can also be a single polypeptide chain. In this alternative instance the single polypeptide chain can in some instances comprise two or more polypeptide subunits fused together to form a protein. The terms “polypeptide” and “protein” also refer to the products of post-expression modifications, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide or protein can be derived from a natural biological source or produced by recombinant technology.

[0059] The terminology used in the present disclosure is for the purpose of describing particular aspects only and is not intended to be limiting.I. Methods for Perfusion Evolution Selection Strategy

[0060] The perfusion-evolved ATF cells of the present disclosure displayed remarkably improved and consistent production capability in both fed-batch and perfusion for various model antibodies examined. Higher cell growth, enhanced viability, and increased productivity with lower lactate level in fed-batch have been observed in the pools generated from the ATF host. Moreover, the clones isolated from the perfusion-evolved ATF host derived pools demonstrated improved outgrowth in 384-well plates, enrichment of high producer population, and improved titer and specific productivity in fed-batch together with consistent phenotypic stability over many generations. Most importantly, increased volumetric and specific productivities with improved product quality demonstrated by the ATF clones in HIP bioreactors suggest that the ATF host is better equipped to deal with the challenges of development ability and manufacture of biotherapuetics in the perfusion environment. Given these remarkable improvements inATFHOST- 100-PCT01 -NPcellular performance, the perfusion-evolved ATF host cell can serve as a cost-effective alternative tool for the costly fed-batch process for production of recombinant proteins of various molecular formats.

[0061] A suitable cell retention device is critical for operating a successful perfusion process, and the use of hollow fiber membranes like ATF and tangential flow filtration (TFF) to retain cells based on size is the most common option. However, one of the main disadvantages of the hollow fiber-based perfusion culture setting is the mechanical shear stress associated with the passage of the cells in the hollow fiber lumens, with the TFF known to introduce more shear than ATF. Since high shear or mechanical damage can be detrimental to cell growth and viability, it is desirable that significant shear stress be avoided to achieve high cell density and maintain high productivity in the perfusion platform. The perfusion-evolved ATF host cell has proved to be a suitable tool to overcome this challenge. The improved performance of the ATF host derived clones in TFF based perfusion environment indicates that these clones are more resistant to TFF induced shear damage compared to the parental CHO clones.

[0062] Intracellular proteomics profiling of the two host types identified differential expression of key pathway proteins that regulate the kinetics of cell growth, cytoskeletal organization, and cellular metabolism. Previous studies have revealed high cell density and high shear stress triggering altered cell development and morphogenesis together with cytoskeletal reorganization (Zamani et al., 2018, Zhan.. Diane Hatton et al, 2020). Therefore, it was conceivable that the directed evolution- sei ection strategy to evolve the CHO host under multiple stresses including the stresses observed in a high-density perfusion culture such as low nutrient levels, low glucose levels, shear stress, low cell specific nutrient, and high metabolic waste levels resulted in a robust, high performing host phenotype better suited for intensified upstream processes.

[0063] Here is presented an innovative approach to create a novel shear-resistant perfusion-friendly CHO host that exhibits improved functional attributes which impact biomanufacturing processes. Insights are provided into the altered intracellular environment in the new host and demonstrate its importance in production performance. The findings indicate that the perfusion-evolved host achieved a requisite phenotype that is contributing to the significant improvement in its manufacturing capability in the perfusion setting. This host does not only enable accelerated early-stage process development; with the alignment of early cell line development with next generation manufacturing platforms, it will simplify the process ofATFHOST- 100-PCT01 -NPselection of the most desirable recombinant cell line allowing delivery of higher quantities of superior drug products to patients.

[0064] Perfusion is an emerging cultivation mode in biomanufacturing that enables significant intensification capacity to help meet demand. (Xu S. et al., BiotechnoL Prog.

[2017] Jul;33(4):867-878.) Culturing cells in perfusion mode allows for a continuous replenishment of the culture medium while the cells are retained inside the bioreactor, thus permitting a more favorable environment that contributes to improved cell metabolism and growth therefore leading to long-term production with high volumetric yield and consistent product quality (Gomez N. et al., J BiotechnoL

[2019] Oct 10;304:70-77 Sinharoy P. et al, Sci Rep.

[2020] Oct 6; 10(1): 16620.) However, perfusion cell culture in production bioreactors has not been widely applied for the manufacture of protein products because of the complex process control, large volumes of perfusion media, and challenges for process development and characterization. (Bielser J.M. et al,

[2018] BiotechnoL Adv. 36(4): 1328-1340; CroughanMS et al,

[2015] BiotechnoL Bioeng. 2015;l 12(4), pp. 648-651.)

[0065] CHO cell lines are derived from the ovary of the Chinese hamster (Cricetulus griseus) ovary cells. (Tijo, J.H., and Puck T. T. (1958) J. Exp. Med. 108(2), pp. 259-268.) CHO cells have been incredibly useful for cell development and cell line technology and are considered an ideal system for expressing recombinant proteins, due to their post-translational protein modification system being similar to that of human cells. Today, they are the most widely used mammalian cells for transfection, expression, and large-scale recombinant protein production, with recombinant manufacturing with CHO cells presenting more than 70% of the entire biopharmaceutical industry. (K.P. Jayapal et al.,

[2007] Chem. Eng. Prog, 103(7): 40-47.) CHO cells have a well-characterized approval history for production of clinical grade materials such as recombinant antibody therapeutics.

[0066] The present disclosure provides methods for a perfusion based directed evolution selection strategy used for the development of a novel ATF host cell line better suited for cell line development and intensified continuous upstream processes, comprising(i) seeding the unevolved CAT-S cells in an alternating tangential flow (ATF) filtration perfusion bioreactor in a glucose-containing cell culture medium;(ii) perfusing the CAT-S cells at a rate to limit the amount of glucose available to the cells such that all glucose added was consumed between days 9 and 14 of the culture;(iii) bleeding the cells to maintain a cellular concentration of 5 x 107cells / mL;ATFHOST- 100-PCT01 -NP(iv) culturing the CAT-S cells for a total of 30 days wherein the cell density exceeded 1.30 x 108cells / mL and the glucose levels in the bioreactor were depleted; and(v) harvesting the perfusion-evolved cells.

[0067] The methods of the present disclosure can be performed in a variety of vessel types or bioreactor types. In some aspects, the term “bioreactor” refers to a bioreactor having a volume of IL, 2L, 5L, 10L, or higher, e.g., about 20L, about 25L, and 50L, about 100L, about 500L, about lOOOL, about 5000L, or any commercially available volume.

[0068] In some aspects, the perfusion bioreactor tank 0.5-10L. In another aspect, the perfusion bioreactor tank is 50-200L. In another aspect, the perfusion bioreactor tank is 500-5000L.

[0069] The methods of the present disclosure involve a perfusion culture. Several cell retention methods are available for perfusion cultures, one of which is tangential flow filtration (TFF). In one aspect, the tangential flow filtration (TFF) is alternating tangential flow filtration (ATF). The term “alternating tangential flow” as used herein refers to a flow arrangement in which a tangential flow travels back and forth along the membrane surface of a hollow-fiber filter and another flow travels in a direction substantially perpendicular to said filter surface. An alternating tangential flow can be achieved according to methods known to the person skilled in the art. Other cell retention methods are contemplated, including tangential flow filtration (TFF), as one of skill in the art would understand.

[0070] In some aspects, perfusion culture and cell concentrations are conducted using a single-use devise, for example, a REPLIGEN™ ATF system. In some aspects, the REPLIGEN™ ATF system is an Xcell ATF® 1 system (0.5L to 2L suspension culture volume), Xcell ATF® 2 (2L to 10L suspension culture volume), Xcell ATF® 4 system (10L to 50L suspension culture volume), Xcell ATF® 6 system (50L to 200L suspension culture volume, or Xcell ATF® 10 system (200L to WOOL suspension volume). Similar devices known in the art can be used to implement the methods disclosed herein.

[0071] In some aspects, the ATF2 module consists of stainless-steel hose barbs, clamps and a 0.2pm polyethersulfone hollow fiber filter (Repligen, S04-P20U- 10-N, Lot 20007711) which was connected to a diaphragm (Repligen D2S1) pump housed in a stainless steel chamber. In some aspects, clamps and o-rings maintained sterility in the hollow fiber module and upper diaphragm chamber (cell side).ATFHOST- 100-PCT01 -NP

[0072] In some aspects, component parts that had contact with the cells were made from the following materials: 316L SS, EPDM, Polycarbonate, Polysulfone, Silicone, Viton®, Polyester and PTFE (Repligen). In some aspects, the reusable parts were soaked and cleaned with 1 : 100 CIP100 alkaline detergent (STERIS Life Sciences) for at least 24 hours followed by neutralization with 1:200 CIP 200 acid detergent (STERIS Life Sciences).

[0073] In some aspects, all reactor and ATF2 module components that were in contact with the cells are Animal Component Free (ACF).

[0074] In some aspects, CAT-S host cells were subcultured into M030-vl2 medium supplemented with 6mM GlutaMAX I. In some aspects, these cells were grown for 3 days prior to seeding in the perfusion bioreactor equipped with ATF at a cell density of 3 x 106cells / mL.

[0075] In some aspects, the starting glucose level in the medium is about 1.0 g / L, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, about 5.0 g / L, about 5.5 g / L, about 6.0 g / L, about 6.5 g / L, about 7.0 g / L, about 7.5 g / L, about 8.0 g / L, about 8.5 g / L, about 9.0 g / L, about 9.5 g / L, about 10.0 g / L. In some aspects, the starting glucose level in the medium is about 4.3 g / L (Figure ID).

[0076] One aspect of the methods disclosed therein is that the perfusion must be tuned so that the available glucose levels in the media are depleted post inoculation. In some aspects, the glucose levels are depleted on post inoculation day 9. In some aspects, the glucose levels are depleted on post inoculation day 10. In some aspects, the glucose levels are depleted on post inoculation day 11. In some aspects, the glucose levels are depleted on post inoculation day 12. In some aspects, the glucose levels are depleted on post inoculation day 13. In some aspects, the glucose levels are depleted on post inoculation day 14. Other nutrients are contemplated as measurable indicators of nutrient depletion during the culture, as one of skill in the art would understand.

[0077] In some aspects, as viable cell density (VCD) and viability starts to decline, cell bleeding is initiated. In some aspects, cell bleeding is initiated on post inoculation day 9. In some aspects, cell bleeding is initiated on post inoculation day 10. In some aspects, cell bleeding is initiated on post inoculation day 11. In some aspects, cell bleeding is initiated on post inoculation day 12. In some aspects, cell bleeding is initiated on post inoculation day 13. In some aspects, cell bleeding is initiated on post inoculation day 14.

[0078] In some aspects, cell bleeding targets a steady concentration of 5*107cells / mL.ATFHOST- 100-PCT01 -NP

[0079] In some aspects, the resulting perfusion-evolved cells are harvested at 7*107cells / mL VCD and 90% viability.

[0080] In some aspects, the perfusion-evolved cells are harvested on day 30. In some aspects, the perfusion-evolved cells are harvested on day 25. In some aspects, the perfusion-evolved cells are harvested on day 26. In some aspects, the perfusion-evolved cells are harvested on day 27. In some aspects, the perfusion-evolved cells are harvested on day 28. In some aspects, the perfusion-evolved cells are harvested on day 29. In some aspects, the perfusion-evolved cells are harvested on day 31. In some aspects, the perfusion-evolved cells are harvested on day 32. In some aspects, the perfusion-evolved cells are harvested on day 33. In some aspects, the perfusion-evolved cells are harvested on day 34. In some aspects, the perfusion-evolved cells are harvested on day 35.

[0081] In some aspects, the harvested perfusion-evolved cells are expanded by culturing in a shake flask. In some aspects, the shake flask volume is 125 mL. In some aspects, the shake flask volume is 250 mL. In some aspects, the shake flask volume is 500 mL. In some aspects, the shake flask volume is 1,000 mL. In some aspects, the shake flask volume is 2,000 mL. In some aspects, the shake flask volume is 2,800 mL.

[0082] In some aspects, the bioreactor, e.g., a high-throughput bioreactor such as an AMBR®15 comprises at least one integrated cell culture analyzer. In some aspects, the cell culture analyzer is a pH measurement module, e.g., an AMBR® Analysis module.II. Perfusion-Evolved ATE Host Cell

[0083] CHO Cell Characteristics

[0084] According to one aspect of the present disclosure, there is provided a perfusion-evolved Chinese Hamster Ovary (CHO) CAT-S Cell having increased specific cellular productivity relative to an unevolved CAT-S cell. As used herein, “perfusion-evolved” comprises subjecting the cell to culturing in an alternating tangential flow (ATF) filtration perfusion bioreactor under high cell volume and nutrient depleted conditions.

[0085] In some aspects, the methods disclosed herein produced a perfusion-evolved CHO CAT-S cell having an improved recombinant antibody yield by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% relative to the parental CHO cell.ATFHOST- 100-PCT01 -NP

[0086] In some aspects, the perfusion-evolved CHO CAT-S cell exhibits increased specific cellular productivity relative to the parental CHO cell. In some aspects, the specific cellular productivity is increased at least 1.2 fold. In some aspects, the specific cellular productivity is increased at least 1.3 fold. In some aspects, the specific cellular productivity is increased at least 1.4 fold. In some aspects, the specific cellular productivity is increased at least 1.5 fold. In some aspects, the specific cellular productivity is increased at least 2.0 fold. In some aspects, the specific cellular productivity is increased at least 2.25 fold. In some aspects, the specific cellular productivity is increased at least 2.5 fold. In some aspects, the specific cellular productivity is increased at least 2.75 fold. In some aspects, the specific cellular productivity is increased at least 3.0 fold. In some aspects, the specific cellular productivity is increased at least 3.25 fold. In some aspects, the specific cellular productivity is increased at least 3.5 fold. In some aspects, the specific cellular productivity is increased at least 3.75 fold. In some aspects, the specific cellular productivity is increased at least 4.0 fold. In some aspects, the specific cellular productivity is increased at least 5.0 fold. In some aspects, the specific cellular productivity is increased at least 7.5 fold. In some aspects, the specific cellular productivity is increased at least 10 fold.

[0087] In some aspects, the polypeptides produced by the perfusion-evolved CAT-S cell are antibodies. Antibodies can include, by way of non-limiting example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, and the like.

[0088] In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of one or more proteins that regulate the kinetics of cell growth compared to the parental CAT-S.

[0089] In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of one or more proteins that regulate cytoskeletal organization compared to the parental CAT-S.ATFHOST- 100-PCT01 -NP

[0090] In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of one or more proteins that regulate cellular metabolism compared to the parental CAT-S.

[0091] In some aspects, the perfusion-evolved CHO CAT-S cell exhibits differential expression of glutathione-S-transf erase compared to the parental CAT-S.

[0092] In some aspects, the perfusion-evolved CHO CAT-S cell produces lower levels of lactate compared to an unevolved CAT-S cell.EXAMPLESBiological Deposit

[0093] The CATS cell line of the disclosure was deposited on March 5, 2024 with the American Type Culture Collection (ATCC) under deposit number PTA-127740. ATCC is located at the following address: Historic District, 10801 University Blvd, Manassas, VA, 20110.Material & MethodsCell culture and stable transfections

[0094] A suspension-adapted CHO cell line derived from CHO-K1 and a glutamine synthetase selection system was used. Stable transfectant pools were generated by nucleofection of linearized expression plasmids and then selected and maintained in medium supplemented with 75 pM methionine sulfoximine (Sigma-Aldrich, MO), and 50 mg / L dextran sulfate (Sigma-Aldrich). Suspension cell cultures were grown at 120 rpm on an orbital shaking platform in a humidified incubator set at 37°C and 6% CO2. Cells were passaged every 3-4 days. Measurement of viable cell density and viability was accomplished using Trypan Blue and a ViCell automated cell counter (Beckman Coulter, CA).Perfusion Host Evolution process in bioreactor

[0095] CHO host cells were cultured in duplicate 3 liter glass stirred tank bioreactors in perfusion process equipped with ATF as the cell retention method. Cells were cultured at set points representative of large-scale manufacturing processes. Temperature of 35.5°C, 300 rpm agitation rate, 50% dissolved oxygen and 7.0+0 .1 pH were monitored and regulated using DASGIP controller software (Eppendorf, Germany). Media and feed were added to each bioreactor during the run. Fresh cell samples were collected every day for subsequent process analysis and biochemical assays. Offline pH, gases (pCO2 and O2), lactate and glucose levelsATFHOST- 100-PCT01 -NPwere analyzed using RAPIDPOINT 500 Blood Gas Analyzer (Siemens, PA). Viable cell density and cell viability were determined using a Vi-CELL. Osmolality was measured using an osmometer (Advanced Instruments Inc., MA).Perfusion process in AMBR250

[0096] CHO host cells were cultured in duplicate ambr250ht perfusion TFF bioreactors within the automated ambr250 system (Sartorius, UK). Temperature of 35.5°C, 300 rpm agitation rate, 50% dissolved oxygen and 7.0+0 .1 pH were monitored and regulated. Proprietary media and feed were added to each bioreactor during the run. Fresh cell samples were collected every day for subsequent process analysis and biochemical assays. Offline pH, gases (pCO2 and pO2), lactate and glucose levels, viable cell density, cell viability, and osmolality were analyzed using a BioProfile FLEX2 (Nova Biomedical, MA).Single cell deposition

[0097] Single cell deposition for recombinant clone generation were performed either using an Influx cell sorter (BD Biosciences, CA) as described previously (Chakrabarti et al 2019) or by Single Cell Printer (SCP, Cytena, Germany). Briefly, 1 x 106cells were sorted by depositing one cell per well into individual wells of 384-well plates containing conditioned medium (Krista Evans, 2015). All plates were incubated at 37°C in a humidified atmosphere with 6% CO2 for outgrowth.Colony outgrowth measurement

[0098] Confluence per well in 384-well plates was measured using a Cellavista imager (Synentec, Germany) and results analyzed using Nyone software (Synentec, Germany).Fed-batch culture at small scale

[0099] Antibody production was evaluated by fed-batch culture in 125 mL Erlenmeyer flasks or 96 deep well (96DW) plates. The production cultures grew at 35.5°C in a humidified 6% CO2 atmosphere for 14 days unless otherwise mentioned. Shaker speed was maintained at 120 rpm for flasks and 350 rpm for 96DW. Cell density and viability were monitored during cultivation in flasks, but not for the 96DW cultures. Proprietary feed was added periodically to the production cultures. Antibody titers in the culture supernatant were determined using ProteinATFHOST- 100-PCT01 -NPA biosensors in an Octet QK384 (Pall ForteBio, Fremont, CA) for 96DW and by HPLC (Agilent Technologies, CA) for flask cultures.Intracellular staining for antibody expression level

[0100] Intracellular expression of the heavy and light chains of antibody molecules was determined by staining cells with fluorescently labeled antibodies specific for heavy or light chains. Briefly, the cells were centrifuged, washed with FACS buffer (1% fetal bovine serum in PBS), and fixed with Fixation Medium A (Thermo Fisher Scientific) for 15 minutes at room temperature. Next, the cells were washed with FACS buffer and stained for 15 minutes at room temperature with the staining solution comprised of goat anti-human IgG (Fc)-Alexa Fluor 488 (Thermo Fisher Scientific) and goat anti-human kappa-APC (Biolegend, CA) in Permeabilization Medium B (Thermo Fisher Scientific). The stained cells were washed and resuspended in FACS buffer before analyzing in a BD Symphony cytometer for the APC and AF488 double positive population. Data analysis was performed using FlowJo software.Proteomic analysis

[0101] Sample Preparation:

[0102] For the sample preparation for LC-MS analysis, CHO (n=5) and ATF (n=4) host cells were included for both data-dependent acquisition (DDA) and data-independent acquisition (DIA) analysis on a timsTOF Pro mass spectrometer (Bruker). Briefly, the cells were resuspended in S-trap lysis buffer consisting of 5% SDS and 50 mM triethylammonium bicarbonate (TEAB) buffer at a pH of 7.55. The solubilization of the cell suspensions was achieved by employing a Retsch mill (MM400) bead beater operating at a frequency of 30 Hz for a duration of 2 minutes. The protein concentration in the lysates was determined using a BCA assay kit from Thermo Fisher.Protein Digestion

[0103] For preparing tryptic peptides from samples, 100 pg of protein lysates was employed using the S-Trap 96-well plate digestion method from Protifi.com, following the manufacturer's protocol as described by HaileMariam et al. (2018). Initially, the proteins in the lysates were reduced with 20 mM tris(2-carboxy ethyl) phosphine at 60°C for a duration of 15 minutes. Following this step, alkylation was performed by treating the samples with 80 mMATFHOST- 100-PCT01 -NPiodoacetamide for 1 hour at room temperature. For protein digestion, mass spectrometry-grade trypsin / lys-C (Promega) was used and digestion were carried out for 2 hours at a temperature of 47°C.

[0104] Following digestion, the peptides were eluted using a series of buffers. Initially, the peptides were eluted with 50 mM TEAB buffer. Then, elution was performed using 0.2% formic acid (FA) in water, followed by elution with a mixture of 60% acetonitrile and 40% water containing 0.2% FA. The eluted peptides were dried and subsequently reconstituted in 0.15% FA in water. Peptide concentration in the sample was measured using Nanodrop eight at 220 nm before the LC-MS analysis. In a separate experiment, 5% of peptides from all cells were pooled and fractionated into 8 fractions using Thermo Fisher basic pH fractionation kit (catalog # 84868).LC-MS / MS Analysis

[0105] The LC-MS / MS analysis was performed using a timsTOF Pro mass spectrometer manufactured by Bruker, coupled with an Evosep One LC system from Evosep Biosystems (source: https: / / pubmed.ncbi.nlm.nih.gov / 30104208 / ), and a nano-electrospray ion source (CaptiveSpray Source, Bruker). The procedure involved loading samples (250 ng peptides) onto a disposable Evotip Cl 8 trap column, following the manufacturer's instructions. The separation of peptides carried out on a 15 cm x 75 pm, 1.9 ReproSil C18 column obtained from PepSep.com. The column was maintained at a temperature of 50°C. To separate the peptides, a gradient of solvent A (0.15% formic acid in water) and solvent B (0.15% formic acid in acetonitrile) was used. This gradient was generated by applying a preset 44 min Evosep LC gradient method.

[0106] For the data-dependent acquisition (DDA) process, the mass spectrometer operated in PASEF mode, with a duty cycle close to 100%. The mass spectrometer recorded spectra ranging from 100 to 1600 m / z. Additionally, ion mobility scan range was 0.85 to 1.30 Vs / cm2with a ramp time of 100 ms. The total duty cycle time for this process was 1.15 seconds. The collision energy increased linearly from 27 to 45 eV in correlation with the ion mobility. Precursors that reached a target intensity of 20,000 units were actively excluded for 0.4 minutes.

[0107] In the data-independent acquisition (DIA)-PASEF mode, the instrument followed a specific scheme. It consisted of two rows, each containing 24 windows. Each row performed eight PASEF scans, and each scan had four steps. The isolation width for each window was set toATFHOST- 100-PCT01 -NP25 m / z. The collision energy was ramped linearly from 20 to 52 eV, depending on the ion mobility. The mass scan range for the DIA-PASEF settings was m / z 400-1200 Da, while the mobility range was 0.57-1.47 Vs / cm2.tims-TOF MS data analysis

[0108] To generate a comprehensive spectral library for the DIA analysis, a hybrid library was created that contained MS data of samples analysed in DDA mode and followed DIA analysis with technical replicates. The combined DDA and DIA acquisition raw files were analysed via Spectronaut (Biognosys AG) software with Pulsar search engine(SN15.6.211220.50606) to build the library using Uniprot Cricetulus Griseus Feb2022 (56342 entries). The search parameters were set as default but included an additional deamidation (NQ) in variable modifications. DIA files (28 files) were processed via Spectronaut using the default settings with precursor and protein FDR cut-off set to 0.01, quantification data filtering set to Q-value 0.5 percentile with global imputing, and cross run normalization strategy set to local normalization.

[0109] The mass spectrometry proteomics data will be made available through ProteomeXchange Consortium via the PRIDE (Perez-Riverol, Y., et al., 2022) partner repository with the dataset identifiers number.Proteomics data results

[0110] A comprehensive proteomic profile of CHO and ATF hosts was generated using DIA-PASEF method, with 77,670 peptides being identified and quantitated, and matched to more than 8000 protein groups. Peptides were quantitated between the two host samples using MaxLFQ label free quantitation method. To find differentially expressed proteins, a statistical analysis including false discovery rate (FDR)-adjusted p-values were calculated was carried out with Perseus software.Statistical analysis[OHl] Data was presented as mean ± standard deviation (SD). Comparisons of mean differences between groups were made by unpaired two-tailed Student’s t-test unless otherwise stated. A probability level of p<0.05 was considered to be statistically significant.ATFHOST- 100-PCT01 -NPGeneration of a novel CHO host cell line by perfusion evolution

[0112] Leveraging the known ability of CHO cells to adapt to different culture conditions, in-house CHO host cells were evolved in perfusion culture under high cell density and nutrient depleted environment (Figure 1 A). First, the cells were grown in a non-perfusion batch culture (Yongky, A., Xu, J., Tian, J., Oliveira, C., Zhao, J., McFarland, K., ... Li, Z. J. (2019). Process intensification in fed-batch production bioreactors using non-perfusion seed cultures. mAbs, 11(8), 1502-1514) for 3 days prior to seeding in the perfusion bioreactor equipped with ATF at a cell density of 3 x 106cells / mL. Perfusion was initiated 2 days after inoculation at 1.2 VVD (volume of media per bioreactor volume per day) and continued until cell harvest. The cells were allowed to grow at high cell density reaching 1.86x108 cells / mL at day 10 with viability close to 90% (Figure IB, C). Perfusion rate was maintained to limit the amount of nutrients available to the cells such that all of the glucose added was consumed and cells experienced glucose depletion between days 9 and 14 (Figure ID). The surviving cells were expected to be those that utilized nutrients most efficiently for proliferation and other lifesustaining functions. As the viable cell density (VCD) and viability started to drop, cell bleeding was initiated at day 15 to keep a steady concentration. After 30 days in perfusion culture, the resulting perfusion-evolved ATF host cells at 7 x 107cells / mL VCD and 90% viability were harvested and transferred to a shake flask for subculture and expansion.Superior performance of the perfusion-evolved host in stable transfectant pools

[0113] To evaluate the performance of the ATF host, the parental CHO host and the perfusion host were transfected with expression plasmids encoding four different format biotherapeutics : three easy-to-express (ETE) antibodies (AZE1, AZE2 and AZE3) and a difficult-to-express (DTE) bispecific antibody (AZDI). The resulting stable transfectant pools were evaluated for fed-batch productivity in shake flasks. For all four molecules, the pools derived from the ATF host displayed higher VCD (Figure 2A-D) and higher viability than the parental CHO pools (Figure 2E-H). The ATF host derived pools also demonstrated a 1.5, 1.3 and 3-fold increase in titer for AZE1 (Figure 3A; 2.3 vs. 3.44 g / L, p=0.21), AZE2 (Figure 3B; 3.4 vs.4.3 g / L, p=0.11) and AZE3 (Figure 3C; 0.76 vs. 2.42 g / L, p=0.0003), respectively, compared to the CHO pools. For DTE molecule AZDI, the ATF pool had a 2.5-fold increase in final titer compared to the CHO pool (Figure 3D; 0.56 vs. 1.4 g / L, p=0.0005). The lactate production (pgATFHOST- 100-PCT01 -NPper cell per day) throughout the culture period found to be lower in the ATF pools than the corresponding CHO pools (Figure 3E-H).

[0114] Intracellular heavy chain (HC) and light chain (LC) protein expression in the pools were investigated to assess whether differences in the percentage distribution of expressing populations could be the cause of the difference in productivity observed between the two hosts. For AZE1 and AZE2, both host derived pools exhibit similar expression of HC and LC, with 89% HC+LC+ for the CHO and 92% for the ATF host and >99% for CHO and ATF pools of AZE3 (Figure 11). Likewise, for AZDI, the analysis revealed similar expression of HC and LC in both hosts, with 89% HC+LC+ for the CHO and 86% for the ATF host (Figure 11). Therefore, the significant difference in productivity of the pools observed between the two hosts is not due to difference in homogeneity of the expressing populations rather due to the improved capability of the ATF host.Evaluation of the perfusion-evolved host in the cell line development process

[0115] Generation of clonal manufacturing cell lines is a crucial step towards ensuring reproducible cell culture performance and generating consistent product quality for biopharmaceuticals. To determine the suitability and applicability of the new perfusion-evolved ATF host for successful biomanufacturing, the performance of clones isolated from the stable pools of AZE1 and AZE3 was evaluated. FACS was used for AZE1 and Single Cell Printer (SCP) for AZE3 to deposit single cells into 384-well plates from the transfectant pools generated from the parental CHO and ATF hosts. Improved outgrowth of the clones derived from ATF host was observed for AZE1 (p<0.04) and similar outgrowth observed for AZE3 (Figure 4A and 4B. respectively). Then, the clones were expanded and evaluated in an automated small-scale productivity screen in a 96DW format (Wang B, Albanetti T, Miro-Quesada G, Flack L, Li L, Klover J, Burson K, Evans K, Ivory W, Bowen M, Schoner R, Hawley-Nelson P. High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system. Biotechnol Prog. 2018 Nov;34(6): 1460-1471). Several top producer clones of AZE1 and AZE3 were selected based on the 96DW titer ranking and their performance was evaluated in high throughput AMBR15 cell culture system. The results revealed a remarkable increase in the average titer (Figures 4C) (p<0.0001) and specific productivity (Figures 4E) (p=0.007) achieved for AZE1 clones generated from the ATF host compared to the parental host. Although no significant difference in titer or specific productivity was observed for clones expressing AZE3,ATFHOST- 100-PCT01 -NPthe distribution of the values indicates an increased frequency of higher producing clones being generated from the ATF host (Figure 4B, 4D, and 4F). No significant difference was observed for end of run (EOR) viability and lactate production for either molecules derived from the two host types (Figure 4G- J).

[0116] While it is unlikely that directed evolution of the CHO host in the perfusion process would introduce phenotypic instability into the expressor population beyond what is normally observed, the phenotypic stability of the two highest producing clones for each of AZE1 and AZE3 was examined by analyzing the fed-batch titers and intracellular antibody expression over 75 generations (population doubling levels, PDL). Clones expressing AZE1 and AZE3 derived from the ATF host (El-Al, E1-A2 and E3-A1 and E3-A2 respectively) were assessed after 20 (early PDL) and 75 (late PDL) generations in culture. All clones demonstrated consistent intracellular antibody expression (Figure 5A and 5B) and secreted antibody productivity (Figure 5C and 5D) irrespective of the PDL number.Evaluation of the performance of the perfusion-evolved clones in perfusion environment

[0117] To better understand the perfusion compatibility of the perfusion host-derived clones El-Al and E3-A1 over the parental CHO clones El-Cl and E3-C1, the clones were cultured in AMBR250 culture vessels under perfusion conditions representative of large-scale manufacturing processes. Our data demonstrated that El-Al and E3-A1 have higher titer compared to the parental CHO clones (Figure 6 A, B). The higher VCD observed for El-Al than the parental CHO clone contributed to higher titer but similar cell specific productivity (Figure 6 A, C and E). In contrast, E3-A1 showed similar VCD as the parental CHO clones, but higher cell specific productivity, which contributed to greater titer (Figure 6 B, D and F). Viability during the culture period remained similar between the clones expressing either of the molecules (Figure 6 G, H). Interestingly, both El-Al and E3-A1 showed lower levels of highly mannosylated product than the parental CHO clones (Figure 61, J).Proteomics Analysis

[0118] The phenotypic study was complemented by a systematic comparative investigation of the global functional changes in the ATF host cells by intracellular proteomics profiling. Quantitative proteomics using liquid chromatography (LC)-MS / MS analyses derived from the fractionation of multiplexed tandem mass tag (TMT)-labeled protein digests of CHOATFHOST- 100-PCT01 -NPand ATF host cells resulted in identification of nearly 7700 protein groups (Supplemental Table 1). The abundance levels of the proteins involved in various cellular functions when analyzed and compared between the two host types, 286 proteins were found to be differentially expressed (-log2 difference >0.75) at 0.05 FDR cutoff level (Figure 7A and B). Biological functions associated with the differentially expressed proteins were determined using the functional feature of the Ingenuity pathway analysis (IP A). The proteomics dataset from each host type was further annotated using Uniprot GO annotation, which includes cellular localization and molecular and biological function. The IPA revealed that several of these proteins are involved in the regulation of cellular metabolism, maintenance of cell morphology, assembly and organization, and intracellular cell signaling. The network of differentially expressed proteins from this subset are shown in Figures 8-10. These results allowed us to gain insights into the underlying molecular processes of the ATF host cell influence on production performance.***

[0119] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth on or more but not exemplary aspects of the present disclosure as contemplated by the inventors, and thus are not intended to limit the present disclosure and the appended claims in any way.

[0120] The information incorporated by reference includes the entire contents of the database entry in the most recent version of the database that was publicly available at the time the present application was filed. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

ATFHOST- 100-PCT01 -NPWHAT IS CLAIMED IS:

1. A perfusion-evolved Chinese Hamster Ovary (CHO) CAT-S cell having increased specific cellular productivity relative to an unevolved CAT-S cell, wherein perfusionevolving the CHO CAT-S cell comprises subjecting the cell to culturing in an alternating tangential flow (ATF) filtration or tangential flow filtration (TFF) perfusion bioreactor under high cell density and glucose depleted conditions.

2. The CHO CAT-S cell of claim 1, wherein cell is cultured in an ATF perfusion bioreactor.

3. The CHO CAT-S cell of claim 1, wherein the cell is culture in a TFF perfusion bioreactor.

4. The perfusion-evolved CHO CAT-S cell of claim 1, wherein perfusion-evolving comprises allowing the cell to grow at a high cell density reaching at least 1.5 x 108, 1.6 x 108, 1.7 x 108, or 1.8 x 108cells / mL.

5. The perfusion-evolved CHO CAT-S cell of claim 1-3, wherein perfusion-evolving comprises perfusing the CHO CAT-S cells at a rate to limit the amount of nutrients available to the cells such that all nutrients added were consumed during perfusion.

6. The perfusion-evolved CHO CAT-S cell of claim 5, wherein perfusion-evolving comprises perfusing the CHO CAT-S cells at a rate to limit the amount of glucose available to the cells such that all glucose added was consumed during perfusion.

7. The perfusion-evolved CHO CAT-S cell of any one of claims 1-6, wherein specific cellular productivity in the perfusion-evolved cell is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, or 1.5-fold compared to an unevolved CAT-S cell.ATFHOST- 100-PCT01 -NP8. The perfusion-evolved CHO CAT-S cell of any one of claims 1-7, wherein the perfusion- evolved CHO CAT-S cell exhibits differential expression of one or more proteins that regulate the kinetics of cell growth, cytoskeletal organization, and / or cellular metabolism.

9. The perfusion-evolved CHO CAT-S cell of claim 8, wherein the perfusion-evolved CHO CAT-S cell exhibits differential expression of glutathione-S-transferase.

10. The perfusion-evolved CHO CAT-S cell of any one of claims 1-9, wherein the cell exhibits decreased expression levels of at least one of ZNF131, NDFIP2, SORBS2, NCAM1, GCH4, WIPI1, ROR1, TMEM160 IRS2, RPS18, FN1, RPL4, GPRC5A, P4HA2, ZNF608, PRKRIP1, Ighvl6-1 or PSMC3 relative to expression levels in an unevolved CAT-S cell.

11. The perfusion-evolved CHO CAT-S cell of any one of claims 1-10, wherein the cell exhibits increased expression levels of at least one of RAB6B, CCDC117, DTL, VHL, HY0U1, DBN1, NFIB, MAP1LC3A, KLHL42, GAK, RLF, SPN, TMOD2, PGGT1B, UBE4B, ATAD5, TOE1, SET, RPL31, NOA1, NNMT, ZBTB38, TOE1, RPL10, UBAC2, ATCB, RPL23, UBE20, FAM102B, BRD7, SIPA1L2, MICAL2, AKNAD1, RBFA, PANK3, EVI5, ROGDI, RNFT1, BYSL, or TMEM175 relative to expression levels in an unevolved CAT-S cell.

12. The perfusion-evolved CHO CAT-S cell of any one of claims 1-11, wherein the cell produces lower levels of lactate compared to an unevolved CAT-S cell.

13. A method for generating perfusion-evolved Chinese Hamster Ovary (CHO) CAT-S cells, comprising:i. seeding an unevolved CAT-S cells in an alternating tangential flow (ATF) filtration perfusion bioreactor in a glucose-containing cell culture medium; ii. perfusing the CAT-S cells at a rate to limit the amount of glucose available to the cells such that all glucose added was consumed between days 9 andATFHOST- 100-PCT01 -NPiii. bleeding the cells to maintain a cellular concentration of 5 x 107cells / mL; iv. culturing the CAT-S cells for a total of 30 days wherein the cell density exceeded 1.30 x 108cells / mL and the glucose levels in the bioreactor were depleted; andv. harvesting the perfusion-evolved cells.

14. The method of claim 13, further comprising expanding the harvested perfusion-evolved cells by culturing in a shaker flask.

15. The method of claim 13 or 14, wherein the ATF-perfusion bioreactor volume is between about 0.5-10L.

16. The method of claim 13 or 14, wherein the ATF perfusion bioreactor volume is between about 50-200L.

17. The method of claim 13 or 14, wherein the ATF perfusion bioreactor volume is between about 500-5000L.

18. The method of any one of claims 13-17, wherein the evolved cells are harvested on day 30.

19. The method of any one of claims 13-17, wherein the perfusion-evolved cells were maintained post-cell bleeding at a viable cell density (VCD) of 7 x 107cells / mL and 90% viability.

20. A perfusion-evolved CHO CAT-S cell produced by the method of any one of claims 13- 19.

21. A perfusion-evolved CHO CAT-S cell line deposited with American Type Culture Collection (ATCC) under deposit number PTA-127740.

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