The present disclosure relates to cells and cell lines, e.g., eukaryotic cells and cell lines, for production of a product, e.g., a recombinant protein. The present disclosure also relates to modulation of the protein degradation pathway for improved production of the product, e.g., the recombinant protein.

BACKGROUND

The generation and selection of high-producing recombinant CHO cell lines has been highlighted as a bottleneck in the cell line development process. Thus, there is a need for methods for identifying and generating cell lines that have the capacity for high production of recombinant proteins.

SUMMARY

The present disclosure is based, in part, on the discovery that there is a correlation between the ability of a cell, e.g., an eukaryotic cell, e.g. a mammalian or a cell other than a mammalian cell, to produce a product, e.g., a recombinant polypeptide, and susceptibility to inhibitors of the protein degradation pathway, e.g., proteasome inhibitors, ubiquitin pathway inhibitors, and ERAD inhibitors. Without wishing to be bound by theory, it is believed that inhibitors of the protein degradation pathway can be used to select for high productivity cells, e.g., cells that are capable of producing high yields of a product, e.g., a recombinant polypeptide. Accordingly, disclosed herein are methods and processes for evaluating, selecting, classifying, or identifying cells for producing a product, e.g., a recombinant polypeptide. Also provided herein are methods and processes for making a cell or cell line for producing a product, e.g., a recombinant polypeptide, by identifying or selecting a cell that is capable of high productivity. These methods and processes described herein include contacting the cell with an inhibitor of protein degradation, e.g., inhibits or reduces the activity of the protein degradation pathway. Thus, provided herein are methods for not only identifying better quality of cells, e.g., with higher production capacity, for producing products, e.g., recombinant polypeptides, but also provides cells that produce better quality of products, e.g., recombinant polypeptide products, e.g., monoclonal antibodies and difficult to express proteins, e.g., bispecific molecules.

In one aspect, the present disclosure features a method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide. In one

embodiment, the method comprises:

a) optionally, providing a cell;

b) contacting the cell (or progeny of the cell) with an inhibitor of protein degradation, e.g., a proteasome inhibitor, a ubiquitin pathway inhibitor, or an endoplasmic reticulum associated degradation (ERAD) inhibitor;

c) evaluating the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, ubiquitin pathway inhibitor, or an ERAD inhibitor, on one or more parameters related to cell function, in the cell (or progeny of the cell);

d) optionally, comparing a value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the one or more parameters with a reference value; and

e) optionally, expressing a product, e.g., a recombinant polypeptide from the cell (or progeny of the cell); and

thereby evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells. In one embodiment, the method comprises providing a cell. In one embodiment, the method comprises comparing a value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the one or more parameters with a reference value. In one embodiment, the method comprises expressing a product, e.g., a recombinant polypeptide from the cell (or progeny of the cell).

The products produced by the cells and methods described herein can be a molecule, a nucleic acid, a polypeptide, or any fusion or hybrid thereof. In one embodiment, the product is a non-naturally occurring material or molecule. In another embodiment, the product is a naturally occurring material or molecule. The cells producing the products described herein are engineered or modified to control, e.g., increase the expression, or produce, e.g., encode, the products described herein. In one embodiment, the cells are engineered or modified to comprise an exogenous nucleic acid that controls, e.g., increases, the expression of an endogenously

expressed product. In another embodiment, the cells are engineered or modified to comprise an exogenous nucleic acid that encodes a product, e.g., an endogenously expressed product, a product that is not endogenously produced by the cell, or a non-naturally occurring product.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises evaluating a cell or a progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises classifying a cell or a progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises identifying a cell or a progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises making a cell or a progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises selecting a cell or a progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, a recombinant polypeptide further comprises, e.g., as a part of step (b), culturing the cell (or progeny of the cell) in contact with the inhibitor of protein degradation.

The methods described herein comprise identifying or selecting a cell with improved, e.g., increased, production capacity, e.g., ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide. In one embodiment, the level, amount, or quantity of the product produced by the identified or selected cell is increased, e.g., by 1-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, as compared to the level, amount, or quantity produced by a cell that has not been contacted by the inhibitor of protein degradation.

The method described herein comprise identifying or selecting a cell with improved, e.g., increased, quality of a product, e.g., recombinant polypeptide. In one embodiment, the quality of the product produced by the identified or selected cell is increased, e.g., by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, as compared to quality of the product produced by a cell that has not been contacted by the inhibitor of protein degradation. In one embodiment, the improved quality of the product comprises one or more of the following: an increased level, amount, or proportion of the desired product, e.g., with respect to unwanted iso forms, fragmented, or truncated forms; an increased level, amount, or proportion of properly folded product; an increased level, amount, or proportion of functional, e.g., enzymatically active, product; a decreased level, amount, or proportion of aggregated product; or a decreased level, amount, or proportion of fragmented or unwanted isoforms.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises comparing a value for the effect of the inhibitor of protein degradation, e.g., a proteasome inhibitor, with a reference value. In embodiments wherein the value has a predetermined relationship with the reference value, the method comprises classifying, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for establishing a culture, e.g., a cell bank. In some embodiments, wherein the value meets or exceeds the reference value, the method comprises classifying, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for establishing a culture, e.g., a cell bank or for production of a polypeptide, e.g., a recombinant polypeptide. In embodiments wherein the value has a predetermined relationship with the reference value, the method comprises classifying, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a polypeptide, e.g., a recombinant polypeptide. In embodiments wherein when the value meets or exceeds a reference value, the cell or a progeny cell or population of progeny cells is classified, identified, or selected as a production cell or a progeny cell or population of progeny cells. In some embodiments, wherein the value has a predetermined relationship with

the reference value, the method comprises selecting the cell (or a progeny of the cell), e.g., for making a cell line or population of cells.

In one embodiment, the parameter related to cell function comprises, e.g., is selected from:

i) cell survival,

ii) culture viability,

iii) the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, iv) proteasome activity; or

v) quality of the expressed product, e.g., a polypeptide, e.g., a recombinant polypeptide, e.g., a more homogeneous product.

In one embodiment, the method comprises evaluating more than one parameter. In one embodiment, the method comprises evaluating more than one parameter, and the parameters measured are compared with a reference value.

Also provided herein are methods for establishing a cell line from the cell or a progeny cell of a cell described herein. In one embodiment, the method further comprises culturing the cell to provide a population of cultured cells, e.g., a population of progeny cells. In one embodiment, the method further comprises selecting a cell from the population of cultured cells, e.g., a population of progeny cells. In one embodiment, the method comprises evaluating the selected cell (or progeny of the cell) for a parameter related to cell function, e.g., providing a value for the parameter, e.g., a parameter related to cell function as described herein. In such embodiments wherein the value has a predetermined relationship with the reference value, selecting the cell (or a progeny of the cell), e.g., for production of a polypeptide, e.g., a recombinant polypeptide. In embodiments wherein the value meets or exceeds a reference value, the cell (or a progeny of the cell), is selected as a production cell. In one embodiment, the method further comprises establishing a cell line from said cell.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, further comprises introducing an exogenous nucleic acid into the cell. In one embodiments, the exogenous nucleic acid encodes a product, e.g., a recombinant polypeptide, or an exogenous nucleic acid that controls, e.g., increases, the expression of a product, e.g., an endogenous polypeptide. In one embodiment, the exogenous nucleic acid is introduced after one or more of steps a), b), c) and d). In one embodiment, the exogenous nucleic acid is introduced prior to one or more of steps a), b), c) and d). In one embodiment, the exogenous nucleic acid encodes a polypeptide, e.g., a recombinant polypeptide, e.g. a therapeutic polypeptide or an antibody molecule selected from Table 1 or 2. In one embodiment, the method further comprises introducing one or more additional exogenous nucleic acids, e.g., a second exogenous nucleic acid, into the cell. In one embodiment, the second exogenous nucleic acid is introduced after one or more of steps a), b), c) and d). In one embodiment, the second exogenous nucleic acid is introduced prior to one or more of steps a), b), c) and d). In one embodiment, the second exogenous nucleic acid is introduced after introduction of the first exogenous nucleic acid. In one embodiment, the second exogenous nucleic acid is introduced prior to introduction of the first exogenous nucleic acid. In one embodiment, the second exogenous nucleic acid is introduced simultaneously with the first exogenous nucleic acid. In one embodiment, the second exogenous nucleic acid encodes a selection marker, e.g., glutamine synthase. In one embodiment, the method further comprises introducing an exogenous nucleic acid comprises transfection, electroporation, or transduction.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, further comprises evaluating the cell for a second property, e.g., determining if the cell comprises an exogenous component, e.g., one or more exogenous nucleic acids, e.g., one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell. In one embodiment, the method comprises determining if the cell comprises one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell, comprises MSX selection, MTX selection (e.g., DHFR system), antibiotic selection, yeast growth selection, selection based on color change or surface expression of a marker, selection based on the Selexis system, or selection based on the Catalant system. In one embodiment, antibiotic selection comprises selection for resistance to an antibiotic selected from hygromycin, neomycin (G418), zeocin, puromycin, or blasticidin.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises evaluating the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on a parameter related to cell function, e.g., survival, viability, or the ability to proliferate, or to produce a product, e.g., a polypeptide, e.g., a polypeptide expressed from an exogenous nucleic acid; and determining, e.g., by MSX selection, if the cell comprises an exogenous nucleic acid integrated into the

chromosomal genome of the cell. In one embodiment, wherein responsive to the evaluation in the effect of the inhibitor of protein degradation step and the determining if the cell comprises an exogenous nucleic acid integrated into the chromosomal genome of the cell step, evaluating, classifying, identifying, or selecting, a cell, progeny cell or population of progeny cells.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, further comprises an additional selection step, e.g., selection by FACS, magnetic separation, e.g., magnetic beads, colony picking, microfluidic cell sorting, or microfluidic cell destruction. In embodiments, the selection step comprises determining whether the cell comprises one or more exogenous nucleic acids. In embodiments, the selection step comprises determining whether the cell comprises one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell.

In one embodiment, the method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide, further comprises introducing to the cell an agent that assists in protein folding, e.g., chaperone molecules or small chemical molecules. In one embodiment, the agent that assists in protein folding comprises a nucleic acid encoding a chaperone protein or component of the protein folding pathway, e.g., XBP1, SRP14, BiP/GRP78, PDI, calnexin or cyclophilin B. In one embodiment, the agent that assists in protein folding comprises a small molecule selected from DMSO, glycerol, or PBA.

In another aspect, the present disclosure features a method of making a cell line, or population of cells, comprising identifying a cell by the methods described herein; and culturing the cell, or population of cells, to provide a cell line or population of cells. In another aspect, the present disclosure features a method of making a cell line, or population of cells, comprising

identifying or selecting a cell by the methods described herein; and culturing the cell, or population of cells, to provide a cell line or population of cells.

In another aspect, the present disclosure features a method of making a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprising: providing a cell or cell population made by the methods described herein; culturing the cell or cell population in a culture medium; and, optionally, retrieving the product, e.g., polypeptide, e.g., the recombinant polypeptide, from the cell or medium in which the cell was cultured.

In another aspect, the present disclosure features a cell, progeny cell, or population of progeny cells, evaluated, classified, or selected by any of the methods described herein.

In another aspect, the present disclosure features a cell, progeny cell, or population of progeny cells, made by any of the methods described herein.

In another aspect, the present disclosure features a cell, progeny cell, or population of progeny cells, which can be made by any of the methods described herein.

In another aspect, the present disclosure features a product, e.g., a polypeptide, e.g., a recombinant polypeptide, produced by or producible by any of the methods described herein or any of the cells described herein.

In another aspect, the present disclosure features a preparation comprising a product described herein, e.g., a product, e.g., a polypeptide, e.g., a recombinant polypeptide, produced by, or producible by, any of the methods described herein or any of the cells described herein.

In another aspect, the present disclosure features a mixture comprising a cell described herein and a product described herein, e.g., a polypeptide, e.g., recombinant polypeptide.

In another aspect, the present disclosure features a preparation of medium conditioned by culture of a cell, progeny cell, or population of progeny cells, described herein, e.g., a cell, progeny cell, or population of progeny cells described herein.

In another aspect, the present disclosure features a method of making a cell, or progeny of a cell, that produces a recombinant protein (e.g., a recombinant therapeutic protein, e.g., a recombinant therapeutic antibody), comprising:

a) optionally, providing a cell;

b) culturing the cell or the progeny of the cell in the presence of an exogenous inhibitor of protein degradation, e.g., a proteasome inhibitor, a ubiquitin pathway inhibitor, or an endoplasmic reticulum associated degradation (ERAD) inhibitor (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more passages or for at least 12, 24, 48, 72 hours, or 1, 2, 4, 8, 16, 32 or more weeks);

c) culturing the cell or progeny of the cell in the absence of the exogenous inhibitor of protein degradation (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages or for at least 12, 24, 48, 72 hours);

d) optionally, evaluating the effect of the inhibitor of protein degradation on the level of recombinant protein produced by the cell or the progeny of the cell, e.g., to obtain a value of recombinant protein produced by the cell or the progeny of the cell; and

e) optionally, comparing the value obtained in d) with a reference value, wherein the reference value is the level of recombinant protein produced by a cell of the same type as the cell provided in a) cultured in the absence of the exogenous inhibitor of protein degradation;

thereby making the cell or progeny of cells.

Additional features or embodiments of any of the methods or compositions described herein include one or more of the following:

Inhibitors of Protein Degradation

In embodiments of any of the methods or compositions described herein, the inhibitor of protein degradation is a proteasome inhibitor. In one embodiment, the proteasome inhibitor inhibits or reduces the activity of one or more of the 20S core subunit, the 19S regulatory subunit, the 1 IS regulatory particle, or a chaperone protein that assist proteasome assembly, e.g., Hsm3/S5b, Nas2/p27, Rpnl4/PAAF1, and as6/gankyrin. In one embodiment, the proteasome inhibitor is selected from MG132 (Z-LLL-al, Z-Leu-Leu-Leu-al, Z-Leu-Leu-Leu-CHO), epoxomicin, bortezomib, ixazomib, carfilzomib, disulfiram, CEP-18770, ONX 0912, salinosporamide, LLnV, CEP 1612, lactacystin, PS-341, and eponomicin.

In embodiments of any of the methods or compositions described herein, the inhibitor of protein degradation is an ERAD inhibitor. In one embodiment, the ERAD inhibitor inhibits or reduces the activity of one or more of calnexin/calreticulin, UDP-glucose-glycoprotein glucosyltransferase, ER degradation enhancing a-mannosidase-like protein (EDEM), ER

mannosidase I, Sec61 , CDC48p (VCP/p97), Hrdl , DoalO, Ubc6, Ubcl , Cuel , or Ubc7. In one embodiment, the inhibitor of protein degradation is eeyarestatin I.

In embodiments of any of the methods or compositions described herein, the inhibitor of protein degradation is an ubiquitin pathway inhibitor. In one embodiment, the ubiquitin pathway inhibitor inhibits or reduces the activity of one or more of: El ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, or E3 ubiquitin ligase.

In embodiments of any of the methods described herein, the method further comprises contacting the candidate cell with a second inhibitor of protein degradation, e.g., a proteasome inhibitor, an ERAD inhibitor, or an ubiquitin pathway inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially. In one embodiment, the first inhibitor of protein degradation and second inhibitor of protein degradation are selected from MG132, epoxomicin, or eeyarestatin 1. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is a proteasome inhibitor and the second inhibitor is a proteasome inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is a proteasome inhibitor and the second inhibitor is an ERAD inhibitor or an ubiquitin pathway inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is an ERAD inhibitor and the second inhibitor is an ERAD inhibitor. In embodiments, the first inhibitor is different from the second inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is an ERAD inhibitor and the second inhibitor is an ubiquitin pathway inhibitor or a proteasome inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is an ubiquitin pathway inhibitor and the second inhibitor is an ubiquitin pathway inhibitor. In one embodiment, the cell is contacted with the first and second inhibitor of protein degradation, e.g., concurrently or sequentially, and the first inhibitor is an ubiquitin pathway inhibitor and the second inhibitor is an ERAD or a proteasome inhibitor.

In embodiments of any of the methods described herein, the cell is contacted with a concentration of the inhibitor of protein degradation that is sufficient to reduce culture viability, by about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, 80%, 85%, 90, 95% or more, e.g., as compared to culture viability, before the culture is contacted with the inhibitor or as compared to a culture that is not contacted with the inhibitor. In one embodiment, the concentration of the inhibitor of protein degradation is a concentration at which less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1 %, or 0.05% total cells survive in culture after contact with the inhibitor of protein degradation. In one embodiment, the concentration of the inhibitor of protein degradation is a concentration at which one or more cells continue to proliferate. In one embodiment, the concentration of the inhibitor of protein degradation is less than 0.5 μΜ MG-132. In one embodiment, the concentration of the inhibitor of protein degradation is about 0.0625 μΜ MG-132. In one embodiment, the concentration of the inhibitor of protein degradation is less than 0.05 μΜ epoxomicin. In one embodiment, the concentration of the inhibitor of protein degradation is about 0.025 μΜ epoxomicin. In one embodiment, the concentration of the inhibitor of protein degradation is a low concentration of eeyarestatin I, e.g., 0.1 μΜ. In one embodiment, the concentration of the inhibitor of protein degradation is less than 0.5 μΜ eeyarestatin I, e.g., about 0.1 μΜ eeyarestatin I.

In embodiments of any of the methods described herein, the cell is contacted by the inhibitor of protein degradation for 24, 48, 72, 96, or 168 hours. In embodiments of any of the methods described herein, the cell (or the progeny of the cell) is cultured in the presence of the inhibitor of protein degradation for 24, 48, 72, 96, or more hours. In embodiments of any of the methods described herein, the cell (or the progeny of the cell) is cultured in the presence of the inhibitor of protein degradation for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages or for at least 12, 24, 48, 72 hours. In embodiments of any of the methods described herein, the cell is contacted by the inhibitor of protein degradation for 24, 48, 72, 96, or 168 hours after a selection step, e.g., MSX selection. In embodiments of any of the methods described herein, the cell is contacted by the inhibitor of protein degradation for 24, 48, 72, 96, or 168 hours prior to a selection step, e.g., MSX selection. In embodiments of any of the methods described herein, the cell is contacted by the inhibitor of protein degradation simultaneously or concurrently with a selection step, e.g., MSX selection.

Cells

In some embodiments of any of the methods or compositions described herein, the method comprises providing a cell that expresses the polypeptide, e.g., the recombinant polypeptide, e.g., a cell that comprises an exogenous nucleic acid that encodes the product, e.g., the polypeptide, e.g., the recombinant polypeptide or controls the expression of the polypeptide, e.g., the recombinant polypeptide.

In embodiments of any of the methods or compositions described herein, the cell comprises an exogenous nucleic acid that encodes the product, e.g., the polypeptide, e.g., the recombinant polypeptide or controls, e.g., increases, the expression of the polypeptide, e.g., the recombinant polypeptide. In embodiments, the exogenous nucleic acid comprises a nucleic acid sequence encoding one or more, e.g., two, recombinant polypeptides. In one embodiment, the cell is of a type that that has been used commercially to produce a polypeptide product, that has been used to produce a polypeptide for pre-clinical work, or a cell that has been used to produce a product, e.g., a polypeptide, for a clinical trial. In one embodiment, the cell expresses a polypeptide, e.g., the recombinant polypeptide, is selected from Table 1 or 2.

In embodiments of any of the methods or compositions described herein, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is from mouse, rat, Chinese hamster, Syrian hamster, monkey, ape (e.g., including human), dog, horse, camel, ferret, or cat. In one embodiment, the cell is a CHO cell, e.g., CHOK1,

CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1 SV GS-KO, CHOS, CHO DG44, CHO DXBl 1 , CHOZN, or a CHO-derived cell. In one embodiment, the cell is a selected from HeLa, HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK, VERO, SP2/0, NSO, YB2/0, EB66, C127, L cell, COS, e.g., COS1 and COS7, QCl -3, CHOK1, CHOK1SV, Potelligent CHOK1 SV, CHO GS knockout, CHOK1 SV GS-KO, CHOS, CHO DG44, CHO DXBl l . and CHOZN.

In embodiments of any of the methods or compositions described herein, the cell is a eukaryotic cell other than a mammalian cell. In one embodiment, the cell is from an insect, plant, duck, parrot, fish, yeast, or fungus.

Products

As described herein, the products produced by the cells and methods of the present disclosure can be a molecule, a nucleic acid, a polypeptide, or any fusion or hybrid thereof. In one embodiment, the product is a non-naturally occurring material or molecule. In another embodiment, the product is a naturally occurring material or molecule. The cells producing the products described herein are engineered or modified to control, e.g., increase the expression, or produce, e.g., encode, the products described herein. In one embodiment, the cells are engineered or modified to comprise an exogenous nucleic acid that controls, e.g., increases, the expression of an endogenously expressed product. In another embodiment, the cells are engineered or modified to comprise an exogenous nucleic acid that encodes a product, e.g., an endogenously expressed product, a product that is not endogenously produced by the cell, or a non-naturally occurring product.

In one embodiment, the product, e.g., the polypeptide, e.g., the recombinant polypeptide, is a therapeutic polypeptide, e.g., for administration to subject having a disease or disorder. In one embodiment, the product, e.g., the polypeptide, e.g., the recombinant polypeptide is a diagnostic polypeptide.

In any of the methods or compositions disclosed herein, the product comprises one or more of antibody molecule, a bispecific molecule, a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, or an enzyme.

In one embodiment, the product comprises an antibody molecule, e.g., a full-length antibody or an antibody fragment. In one embodiment, the product comprises an antibody molecule or fragment thereof coupled to, e.g., covalently or non-covalently, a second agent, e.g., a polypeptide, e.g., a toxin. In one embodiment, the antibody molecule is a monoclonal antibody. In one embodiment, the antibody molecule binds to a tumor or cancer-associated antigen. In one embodiment, the antibody molecule binds to tumor associated glycoprotein (TAG72). In one embodiment, the product comprises a bispecific molecule, e.g., provided in Table 2.

In one embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, is a human polypeptide which does not differ in amino acid sequence from a naturally occurring isoform of the human polypeptide.

In another embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, differs from a naturally occurring isoform of the human polypeptide by no more than 1 , 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

In yet another embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, differs from a naturally occurring isoform of the human polypeptide or protein by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of its amino acid residues.

In any of the methods or compositions disclosed herein, the product comprises a polypeptide, e.g., a recombinant polypeptide, selected from Table 1 or Table 2. In one embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, is a polypeptide from Table 1 or Table 2.

In another embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, differs from a polypeptide from Table 1 or Table 2 by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

In yet another embodiment, the product, e.g., polypeptide, e.g., a recombinant polypeptide, differs from a polypeptide from Table 1 or Table 2 by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of its amino acid residues.

In any of the methods or compositions described herein, an exogenous nucleic acid comprising a nucleic acid sequence that controls the expression of a product or encodes a product, e.g., a recombinant polypeptide, is introduced to the cell. In any of the methods or compositions described herein, the cell comprises an exogenous nucleic acid comprising a nucleic acid sequence encoding a product, e.g., a recombinant polypeptide. In one embodiment, the exogenous nucleic acid is integrated into a nucleic acid in the cell, e.g., the chromosomal genome of the cell. In another embodiment, the exogenous nucleic acid is not integrated into the chromosomal genome of the cell, but is integrated on an artificial chromosome or plasmid, e.g., such that the exogenous nucleic acid is maintained in the cell or progeny of the cell.

In any of the methods or compositions described herein, the exogenous nucleic acid may further comprise one or more, e.g., two, three, four, or five, additional nucleic acid sequences, e.g., encoding products or other sequences or polypeptides for controlling the expression of a product.

In any of the methods or compositions described herein, the exogenous nucleic acid comprises a plasmid or a vector, e.g., an expression or viral vector. In one embodiment, the exogenous nucleic acid comprises a glutamine synthetase (GS) expression vector. In one embodiment, the exogenous nucleic acid further comprises a nucleic acid sequence encoding a selection marker. In one embodiment, the selection marker comprises glutamine synthetase, DHFR, or a gene that confers antibiotic resistance. In one embodiment, the nucleic acid sequence encoding the selection marker is operably linked to a first promoter and the nucleic acid sequence encoding the product, e.g., the polypeptide, e.g., the recombinant polypeptide is operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are different. In one embodiment, the promoter operably linked to the selection marker is a weak promoter, e.g., SV40E promoter. In one embodiment, the promoter operably linked to the nucleic acid sequence encoding the polypeptide, e.g., the recombinant polypeptide is a strong promoter, e.g., a CMV promoter, e.g., hCMV-MIE promoter.

In any of the methods described herein, the method comprises expressing the product, e.g., polypeptide, e.g., recombinant polypeptide, from the cell or a progeny cell. In one embodiment, the product, e.g., polypeptide, e.g., recombinant polypeptide, is secreted from the cell or progeny cell, e.g., into the culture medium.

In any of the methods or compositions described herein, the expressed product, e.g., polypeptide, e.g., recombinant polypeptide, is evaluated for a parameter related to a physical or functional property, e.g., primary sequence, glycosylation, primary, secondary, tertiary, or quaternary structure, activity, degree of glycosylation, degree of aggregation, proportion or level of the expressed protein having a preselected property, e.g., having a preselected monomeric, dimeric, or trimeric structure, or the level or proportion of the expressed polypeptide having a preselected structure, e.g., non-denatured or non-aggregated structure. In embodiments, the method comprises providing a value for the parameter. In embodiments, the method comprises comparing a value for the parameter with a reference value.

In embodiments of any of the methods or compositions described herein, the value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the parameter related to cell function exceeds the reference value by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more. In embodiments the value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the parameter related to cell function exceeds the reference value by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold or more. In embodiments, the reference value is the value of the effect of the inhibitor of protein degradation on a parameter related to cell function of a reference cell or the value of the parameter related to cell function of the cell that has not been contacted with the inhibitor of protein degradation. In embodiments, the parameter comprises the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, e.g., a recombinant polypeptide expressed from an exogenous nucleic acid. In embodiments, the parameter comprises the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, and wherein the increase is determined by measuring or quantifying the product, e.g., the polypeptide, e.g., the recombinant polypeptide, in the cell or secreted by the cell. In embodiments, the parameter comprises cell survival, and wherein the increase or decrease is determined by measuring or quantifying the number of apoptotic cells. In embodiments, the parameter comprises culture viability, and wherein the increase or decrease is determined by measuring or quantifying the number live cells.

In embodiments where the recombinant polypeptide is secreted from the cell, the methods can include a step for retrieving, collecting, or separating the recombinant polypeptide from the cell, cell population, or the culture medium in which the cell was cultured.

In certain embodiments of any of the preparations described herein, at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the polypeptides in the preparation are properly folded or functionally active.

In certain embodiments of any of the mixtures described herein,

the product, e.g. the polypeptide, e.g., the recombinant polypeptide, is present at a higher concentration, e.g., at least, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher concentration, by weight or number, than would be seen in a cell that has not been contacted with inhibitor of protein degradation; or

wherein at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the product, e.g. the polypeptides, e.g., the recombinant polypeptides, in the mixture are properly folded or functionally active.

In certain embodiments of any of the preparations of medium described herein, the product, e.g., the polypeptide, e.g., the recombinant polypeptide, is present at a higher concentration, e.g., at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30% higher concentration, by weight or number, than would be seen in a cell that has not been contacted with an inhibitor of protein degradation; or

wherein at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the product e.g., the polypeptides, e.g., the recombinant polypeptides, in the mixture are properly folded or functionally active.

In embodiments, any of the methods described herein can further comprise any of steps (f), (g), or both (f) and (g):

f) culturing the cell, or progeny of the cell, in the absence of the exogenous inhibitor of protein degradation (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages or for at least 12, 24, 48, 72 hours);

g) evaluating the level of recombinant protein produced by the cell, or progeny of the cell, e.g., to obtain a value of recombinant protein produced by the cell or progeny of the cell; and

h) optionally, comparing the value obtained in g) with a reference value, wherein the reference value is the level of recombinant protein produced by a cell of the same type as the cell provided in a) cultured in the absence of the exogenous inhibitor of protein degradation.

In embodiments, e.g., embodiments of the methods of making herein, the method further comprises selecting the cell for use in a method of manufacturing a recombinant protein (e.g., a recombinant therapeutic protein). In embodiments, the method further comprises introducing an exogenous nucleic acid into the cell (e.g., after step c), after step d), after step e) or after step f)), optionally, wherein introducing the exogenous nucleic acid comprises transfection,

electroporation, or transduction.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, sub-headings or numbered or lettered elements, e.g., (a), (b), (i) etc, are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily

discrete from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-1C are a series of graphs that show the initial assessment of the indicated protein degradation inhibitors at various concentrations on recombinant CHO cell line growth parameters. Samples were taken at the indicated timepoints and cell concentrations together with culture viability were determined on a ViCell instrument (%).

Figures 2A-2F are a series of graphs that show further assessment of the indicated protein degradation inhibitors at various concentrations on recombinant CHO cell line growth parameters. Samples were taken at the indicated timepoints and cell concentration together with culture viability was determined on a ViCell instrument (%).

Figures 3A-3D are a series of graphs that show the viable cell concentration and culture viability for a panel of CHO antibody producing cell lines when cultured in the presence of MG132. Mean viable cell concentration and culture viability after culturing for 24 and 48 hours in the presence of 0.5 μΜ MG132 (bars with hatching) or DMSO (bars with dashed hatching). Error bars show the standard deviation of triplicate samples taken at each timepoint.

Figures 4A-4D are a series of graphs that show the viable culture cell concentration and culture viability for a panel of CHO antibody producing cell lines when cultured in the presence of epoxomicin. Mean viable cell concentration and culture viability after culturing for 24 and 48 hours in the presence of 0.05 μΜ epoxomicin (bars with hatching) or DMSO (bars with dashed hatching). Error bars show the standard deviation of triplicate samples taken at each timepoint.

Figures 5A-5D are a series of graphs that show the viable cell concentration and culture viability for a panel of CHO antibody producing cell lines when cultured in the presence of eeyarestatin I. Mean viable cell concentration and culture viability after culturing for 24 and 48 hours in the presence of 10 μΜ eeyarestatin I (bars with hatching) or DMSO (bars with dashed hatching). Error bars show the standard deviation of triplicate samples taken at each timepoint.

Figures 6A-6G are a series of plots that show the correlations between susceptibility (e.g, culture viability and/or viable cell concentration in the presence of inhibitor) to proteasome and ERAD inhibitors and productivity data from fed-batch and bio reactor cultures for the panel of CHO cell lines that produce a monoclonal antibody, at one concentration of the proteasome and EPvAD inhibitors. The data was plotted and the best fit line identified by linear regression analysis. The relationship between the parameters was assessed using the Pearson Product Moment Correlation coefficient. The correlations found to be statistically significant (p<0.05) are shown.

Figures 7A-7F are a series of gel images showing the analysis of proteasome activity using activity profiling probes and fluorescent intensity. Proteasome activity was determined in a range of antibody producing cell lines using fluorescently tagged activity probes with differing reactive groups. Cell lysates incubated with the probes were analyzed on 14% SDS-PAGE and the fluorescence was then measured on a Typhoon 9400 instrument using Cy3 and Cy2 filters. Figures 7A and 7B show the MV151 probe, Figures 7C and 7D the MVB003 probe, and Figures 7E and7F the MVB072 probe. The right hand images (Figures 7B, 7D, and 7F) show Coomassie blue stained images as loading controls.

Figures 8A-8F are a series of images showing the analysis of proteasome activity using activity profiling probes and fluorescent intensity. Proteasome activity was determined in a range of antibody producing cell lines using fluorescently tagged activity probes with differing reactive groups. Cell lysates incubated with the probes were analyzed on 14% SDS-PAGE and the fluorescence was then measured on a Typhoon 9400 using Cy3 and Cy2 filters. Figures 8A and 8B show the RUB1001 probe, Figures 8C and 8D the RUB1018 probe, and Figures 8E and 8F the no probe control. The right hand images (Figures 8B, 8D, and 8F) show Coomassie blue stained images as loading controls.

Figures 9A-9F are a series of graphs showing the activity of the different catalytic subunits of the proteasome as determined using the activity based profiling method and subsequent quantitation of band intensity. Densitometry of the bands from the gels in Figures 7A, 7C, and 7E, 8A, 8C, and 8E was performed using Life Technologies ImageQuant®. Graphs show intensity of the bands for each probes tested as well as the sum of all probes summarized in the total proteasome graph.

Figure 10 is a schema depicting the protocol for cell line construction with the CHOK1 GS-KO cell line. Methionine sulfoximine (MSX) selection at a range of concentrations was performed at 24 hours after transfection of 40 μg of linearized DNA. Proteasome inhibitors were then added at either 24, 96 or 168 hours after transfection at a range of concentrations and combinations following a design of experiments plan.

Figure 11 is a graph showing the growth characteristics of cell populations generated using a cell line construction process with MSX and varying proteasome inhibitor selection pressures. Culture viability and viable cell concentration of the GSKO cell pools expressing the model monoclonal antibody (Antibody A) were determined every 48-72 hours following cell line construction with proteasome inhibitors in addition to MSX.

Figure 12 is a graph showing the antibody concentrations achieved from cell populations generated using a cell line construction process with MSX and varying proteasome inhibitor selection pressures with varying selection pressures. GSKO cell lines expressing Antibody A were cultured under batch culture conditions and an ELISA assay performed with supernatant collected after 192 hours of culture following cell line construction with proteasome inhibitors in addition to MSX selection pressure.

Figure 13 depicts the experimental design to assess the use of exemplary proteasome inhibitors described herein during cell line construction.

Figure 14 is a bar graph depicting the average monoclonal antibody product

concentrations achieved following the recombinant GS-CHO cell line construction process with the various inhibitors in batch cultures setup from cells continually cultured in the presence of the inhibitors. Assessment of cB72.3 monoclonal antibody concentration from supernatant samples taken at 48, 96 and 168 hours of 20 mL batch culture following seeding at 0.2 xlO6 viable cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Product titre was assessed using Protein A sensors on an Octet system (n=3 for 37.5 μΜ MSX control, n=2 for inhibitor treatments except 25 nM

Epoxomicin n = l).

Figure 15 depicts a western blot analysis for monoclonal antibody product achieved following the recombinant GS-CHO cell line construction process with the various inhibitors in batch cultures setup from cells continually cultured in the presence of the inhibitors. Assessment of cB72.3 concentration from supernatant samples taken at 168 hours of culture following seeding at 0.2 xlO6 viable cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Western blots were probed using anti-heavy chain antibody (Sigma).

Figure 16 is a bar graph depicting the average monoclonal antibody product

concentrations achieved following the recombinant GS-CHO cell line construction process with

the various inhibitors in batch cultures setup from cells cultured for two passages in the absence of the inhibitors. Assessment of cB72.3 monoclonal antibody concentration from supernatant samples taken at 48, 96 and 168 hours of 20 mL batch culture following seeding at 0.2 xlO6 viable cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Product titre was assessed using Protein A sensors on an Octet system (n=3 for 37.5μΜ MSX control, n=2 for inhibitor treatments except 25 nM

Epoxomicin n=l).

Figure 17 depicts a western blot analysis for monoclonal antibody product following the recombinant GS-CHO cell line construction process with the various inhibitors in batch cultures setup from cells cultured for two passages in the absence of the inhibitors. Assessment of cB72.3 concentration from supernatant samples taken at 168 hours of culture following seeding at 0.2 xlO6 viable cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Cell pools were passaged twice in the absence of the inhibitors before the sampling batch cultures were run. Western blots were probed using anti-heavy chain antibody (Sigma).

Figure 18 is a bar graph depicting the average specific productivities calculated following the cell line construction process immediately after treatment with inhibitors. Specific productivities were calculated from data collected on viable cell number (determined on the Vicell) and product concentration (derived using Protein A probes on the octet system) from supernatant samples taken at 48, 96 and 168 hours of culture following seeding at 0.2 xlO6 cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Individual specific activities were calculated for each cell pool and then the average for each treatment determined (n=3 for 37.5 μΜ MSX control, n=2 for inhibitor treatments except 25 nM Epoxomicin n=l).

Figure 19 is a bar graph depicting the average cell specific monoclonal antibody productivities calculated achieved following the recombinant GS-CHO cell line construction process with the various inhibitors in batch cultures setup from cells cultured in the absence of the inhibitors for two passages. Specific productivities were calculated from the determined cell numbers (determined on a Vicell instrument) and product concentration (derived using Protein A probes on the octet system) from supernatant samples taken at 48, 96 and 168 hours of batch culture following seeding at 0.2 xlO6 cells/ml of cell pools generated from cell line construction with proteasome inhibitors in addition to MSX selection pressure. Individual specific activities were calculated for each cell pool and then the average for each treatment determined (n=3 for 37.5 μΜ MSX control, n=2 for inhibitor treatments except 25 nM Epoxomicin n=l).

Figure 20 is a bar graph depicting the antibody product concentrations achieved post transient transfection in batch culture of the host cell pools following culture in the presence of either proteasome or ERAD inhibitors. Assessment of cB72.3 concentration from supernatant samples taken at 48, 96 and 168 hours post transfection following electroporation of lxl 07 cells with 20 μg of DNA and resuspension into 20 ml cultures. Prior to electroporation, the host cell line was cultured in the presence of the indicated proteasome and ERAD inhibitors at the concentrations indicated. Product titre was assessed using Protein A sensors on the Octet system.

Figure 21 is a bar graph depicting the antibody product concentrations achieved post transient transfection of CHO host cell pool following initial culture in the presence of either proteasome or ERAD inhibitors and then subsequent subculture for 2 passages in the absence of the inhibitors before the transient batch culture. Assessment of cB72.3 concentration from supernatant samples taken at 48, 96 and 168 hours post transfection following electroporation of lxlO7 cells with 20 μg DNA and resuspension into 20 ml batch cultures. Prior to electroporation the host cell line was initially cultured in the presence of proteasome and ERAD inhibitors, and then passaged twice without the presence of inhibitors. Product titre was assessed using Protein A sensors on the Octet system.

Figure 22 depicts a western blot analysis for antibody in the supernatant of batch cultures post transient transfection of the host cell pools both immediately after culture in the presence of either proteasome or ERAD inhibitors (left gel) or following subculture for two passages in the absence of the inhibitors (right gel). Assessment of cB72.3 concentration from supernatant samples taken at 168 hours post transfection following electroporation of lxlO7 cells with 20 μg DNA and resuspension into 20 ml batch cultures. Prior to electroporation the host cell line was initially cultured in the presence of proteasome and ERAD inhibitors, then passaged twice in the absence of the inhibitors. Western blots were probed using anti-heavy chain antibody (Sigma).

Figure 23 is a bar graph depicting the estimated cell specific productivities determined from supernatant samples taken during routine culture of recombinant cell pools generated using the protein degradation inhibitors at different passage numbers. Assessment of cB72.3 concentration from supernatant samples taken during routine subculture every 3-4 days

following revival of successful pools from the cell line construction process with the addition of proteasome inhibitors. Product titre was assessed using Protein A sensors on the Octet system and then the level of product per cell was determined using cell counts determined on the ViCell.

DETAILED DESCRIPTION

Recombinant proteins or polypeptides can be produced by recombinant DNA technology, expressed by host cells, and can be either purified from the host cell (e.g., E. coli) or secreted into the fluid, e.g., cell medium, in which the host cell is cultured and purified from the fluid. Cells capable of producing recombinant proteins or polypeptides in high yields and of appropriate quality are highly desired in the field. The methods disclosed herein for evaluating, classifying, identifying, making, or selecting a cell for production of a recombinant polypeptide are useful for identifying or making high productivity cells, to obtain high yields of recombinant polypeptide product or to provide higher quality preparations of recombinant polypeptide product. The methods disclosed herein are particularly useful for production of recombinant therapeutic polypeptides, where there is a demand for efficient cell line development, large quantities of the recombinant therapeutic polypeptide product, and high grade of quality for therapeutic use in patients.

DEFINITIONS

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 the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" can mean one cell or more than one cell.

As used interchangeably herein, the terms "inhibitor of protein degradation" or "inhibitor of the protein degradation pathway", refers to an agent, e.g., a compound, which decreases (e.g., slows, attenuates, changes) the degradation of proteins, e.g., the reduction of proteins to their polypeptide, peptide, or amino acid constituents, either partially or fully, during protein synthesis and secretion in the cell. By way of example, an inhibitor of protein degradation can be identified by the ability to prevent or decrease (e.g., slow, attenuate, or change) one or more of the following: identification of a protein for degradation; tagging of a protein for degradation; retrotranslocation of a protein from the ER to the cytosol; association of a protein with the proteasome or association of the proteasome with the protein; entry of a protein to the proteasome; unfolding of a protein for entry into the proteasome; processing of the protein by the proteasome; release of the processed peptides from the proteasome; association of the

proteasome with the endoplasmic reticulum; and/or proteasomal capacity of the cell; e.g., in a cell treated with a candidate inhibitor as compared to a similar cell not treated with the candidate inhibitor. In another embodiment, an inhibitor of protein degradation causes conditions to arise in the cell that prevents or reduces proteasomal activity, e.g., in a cell treated with a candidate inhibitor as compared to a similar cell not treated with the candidate inhibitor. In yet another embodiment, an inhibitor of protein degradation saturates the proteasomal activity of the cell, e.g., in a cell treated with a candidate inhibitor as compared to a similar cell not treated with the candidate inhibitor. Proteasomal degradation of a protein, e.g., an endogenously or exogenously expressed protein, can be measured or quantified by heavy/light isotope pulse-labelling approaches, such as stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry (MS). Additional methodologies available in the art for assessing protein degradation are further described in Alvarez-Castelao et al., Biochemistry Research

International, Volume 2012 (2012), Article ID 823597, 11 pages, hereby incorporated by reference.

As used herein, the term "protein degradation pathway" refers to cellular processes that result in the reduction of proteins to their polypeptide, peptide, or amino acid constituents, either partially or fully, e.g., the degradation of misfolded, surplus, truncated, or nonfunctional proteins. Exemplary elements of the protein degradation pathway include, but are not limited to, a proteasome (e.g. 26S proteasome or component thereof, e.g., 20S core or catalytic subunit, 19S regulatory subunit, or 1 IS regulatory subunit), enzymes that promote the identification of

proteins to be degraded or ubiquitination (e.g., El ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, or E3 ubiquitin ligase); or enzymes that promote the translocation or retrotranslocation of proteins from the ER to another element of the protein degradation pathway, e.g., cytosol or proteasome (e.g., retrotranslocation enzyme/complex).

As used herein, the term "proteasome inhibitor" refers to a molecule that prevents or reduces the activity of a proteasome or a component of the proteasome. In one embodiment, the proteasome comprises a 20S catalytic or proteolytic core subunit, and one or more regulatory subunits, e.g., one or more 19S regulatory particles and/or one or more 1 IS regulatory particles. In embodiments, the activity of a proteasome or a component of the proteasome includes one or more of the following: association of a protein with the proteasome or association of the proteasome with the protein; entry of a protein to the proteasome; unfolding of a protein for entry into the proteasome; processing of the protein by the proteasome (e.g., reduction of the protein to peptide or amino acid constituents, partially or folly); release of the processed peptides or amino acids resulting from the processing of a protein from the proteasome; and association of the proteasome with the endoplasmic reticulum. In one embodiment, the proteasome inhibitor reduces the activity of the proteasome, or of a component of the proteasome, by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the activity of a proteasome or a component thereof in the absence of the inhibitor.

As used herein, the term "ubiquitin pathway inhibitor" refers to a molecule that prevents or reduces the activity of one or more components of the ubiquitin pathway. The ubiquitin pathway is the process by which a target protein to be degraded is identified and tagged with ubiquitin molecules for recognition by the proteasome for degradation. Components of the ubiquitin pathway include ubiquitin activating enzymes (El), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). In embodiments, the activity of one or more components of the ubiquitin pathway includes one or more of the following: identification of a protein for degradation, e.g., by an E3 ubiquitin ligase or substrate identification complex; activation of ubiquitin, e.g., by an El activating enzyme; transfer of the ubiquitin from an El to an E2 enzyme; and conjugation of a ubiquitin to a protein or to a ubiquitin molecule or chain (e.g., elongating a ubiquitin chain) on a protein, e.g, by an E3 ubiquitin ligase; deubiquitination by a deubiquitinating enzyme. In one embodiment, the ubiquitin pathway inhibitor reduces the

activity of the one or more components of the ubiquitin pathway by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, as compared to the activity of one or more components of the ubiquitin pathway in the absence of the ubiquitin pathway inhibitor.

As used herein, the term "ERAD inhibitor" refers to a molecule that prevents or reduces the activity of one or more components of the endoplasmic reticulum associated degradation (ERAD) pathway. The ERAD pathway targets misfolded, mutated, or nonfunctional proteins of the endoplasmic reticulum for ubiquitination and subsequent degradation by the proteasome. Components of the ERAD pathway include chaperone proteins, e.g., Hsp70 family members, Hsp40 family members, Hsp90 family members, nucleotide exchange factors, small heat shock proteins, lectin-like chaperones; enzymes that identify or tag misfolded proteins, e.g., a-manosidases; enzymes or protein complexes that assist in retrotranslocation of misfolded proteins, e.g., retro-translocation complex (RTC); enzymes or protein complexes that ubiquitinate retro -translocated proteins, e.g., Hrd-Der ubiquitin ligase complex; and enzymes or protein complexes that deliver ubiquitinated proteins to the proteasome, e.g., Cdc48-Ufdl-Npl4 complex. In embodiments, the activity of one or more components of the ERAD pathway includes one or more of the following: identification or tagging of a misfolded, non-functional protein for degradation; retrotranslocation of a protein from the ER to the cytosol; ubiquitination of the protein at the ER membrane, e.g., integrated into the ER membrane; delivery of the ubiquitinated protein to the proteasome; or deubiquitination of the proteins. In one embodiment, the ERAD inhibitor reduces the activity of the one or more components of the ERAD pathway by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, as compared to the activity of one or more components of the ERAD pathway in the absence of the ERAD inhibitor.

As used herein, the expression "a low concentration" when used in conjunction with an inhibitor of protein degradation, e.g., "a low concentration of a proteasome inhibitor", "a low concentration of an ubiquitination pathway inhibitor", or "a low concentration of an ERAD inhibitor", refers to a concentration of an inhibitor of protein degradation that results in a reduction in culture viability, e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction in culture viability when treated with the inhibitor of protein degradation, as compared to a culture that has not been treated with the inhibitor of protein degradation. As the purpose of using the inhibitor of protein degradation is to identify or select cells with improved production capacity or improved production quality, a low

concentration of an inhibitor of protein degradation is a concentration that is less than that used to kill diseased cells for therapeutic benefit, e.g., cancer cells. It is understood that a culture viability below 30%, e.g., 20%, 10%, 5%, 1 %, 0.5%, 0.1%, may still result in a population of selected cell that have improved productivity capacity and produce better quality products, and is also within the scope of the present disclosure. In another embodiment, the low concentration of the inhibitor of protein degradation is a concentration that partially, but not completely, inhibits protein degradation, e.g., a protein or process associated with protein degradation. By way of example, a low concentration of a proteasome inhibitor is a concentration that partially, but not completely inhibits protein degradation. Methods for assaying proteasome activity, e.g., inhibition of proteasome activity, are described herein.

As used herein, the term "endogenous" refers to any material from or naturally produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced to or produced outside of an organism, cell, tissue or system. Accordingly, "exogenous nucleic acid" refers to a nucleic acid that is introduced to or produced outside of an organism, cell, tissue or system. In an embodiment, sequences of the exogenous nucleic acid are not naturally produced, or cannot be naturally found, inside the organism, cell, tissue, or system that the exogenous nucleic acid is introduced into. In one embodiment, the sequences of the exogenous nucleic acids are non-naturally occurring sequences, or encode non-naturally occurring products.

As used herein, the term "heterologous" refers to any material from one species, when introduced to an organism, cell, tissue or system from a different species.

As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" are used interchangeably and refers to deoxyribonucleic acid (DNA) or ribonucleic acid (R A), or a combination of a DNA or RNA thereof, and polymers thereof in either single- or double-stranded form. The term "nucleic acid" includes, but is not limited to, a gene, cDNA, or an mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless specifically limited, the term encompasses molecules containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91 -98 (1994)).

As used herein, the terms "peptide," "polypeptide," and "protein" are used

interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. In one embodiment, a protein may comprise of more than one, e.g., two, three, four, five, or more, polypeptides, in which each polypeptide is associated to another by either covalent or non-covalent bonds/interactions. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.

As used herein, "product" refers to a molecule, nucleic acid, polypeptide, or any hybrid thereof, that is produced, e.g., expressed, by a cell which has been modified or engineered to produce the product. In one embodiment, the product is a naturally occurring product or a non-naturally occurring product, e.g., a synthetic product. In one embodiment, a portion of the product is naturally occurring, while another portion of the product is non-naturally occurring. In one embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. In one embodiment, the product is suitable for diagnostic or pre-clinical use. In another embodiment, the product is suitable for therapeutic use, e.g., for treatment of a disease. In one embodiment, the product is selected from Table 1 or Table 2. In one embodiment, the modified or engineered cells comprise an exogenous nucleic acid that controls expression or encodes the product. In other embodiments, the modified or engineered cells comprise other molecules, e.g., that are not nucleic acids, that controls the expression or construction of the product in the cell.

In one embodiment, the modification of the cell comprises the introduction of an exogenous nucleic acid comprising a nucleic acid sequence that controls or alters, e.g., increases, the expression of an endogenous nucleic acid sequence, e.g., endogenous gene. In such embodiments, the modified cell produces an endogenous polypeptide product that is naturally or endogenously expressed by the cell, but the modification increases the production of the product and/or the quality of the product as compared to an unmodified cell, e.g., as compared to endogenous production or quality of the polypeptide.

In another embodiment, the modification of the cell comprises the introduction of an exogenous nucleic acid encoding a recombinant polypeptide as described herein. In such embodiments, the modified cell produces a recombinant polypeptide product that can be naturally occurring or non-naturally occurring. In such embodiments, the modified cell produces a recombinant polypeptide product that can also be endogenously expressed by the cell or not. In embodiments where the recombinant polypeptide product is also endogenously expressed by the cell, the modification increases the production of the product and/or the quality of the product as compared to an unmodified cell, e.g., as compared to endogenous production or quality of the polypeptide.

As used herein, "recombinant polypeptide" or "recombinant protein" refers to a polypeptide that can be produced by a cell described herein. A recombinant polypeptide is one for which at least one nucleotide of the sequence encoding the polypeptide, or at least one nucleotide of a sequence which controls the expression of the polypeptide, was formed by genetic engineering (of the cell or of a precursor cell); e.g., at least one nucleotide was altered, e.g., it was introduced into the cell or it is the product of a genetically engineered rearrangement. In an embodiment, the sequence of a recombinant polypeptide does not differ from a naturally occurring isoform of the polypeptide or protein. In an embodiment, the amino acid sequence of the recombinant polypeptide differs from the sequence of a naturally occurring isoform of the polypeptide or protein. In an embodiment, the recombinant polypeptide and the cell are from the same species. In an embodiment, the recombinant polypeptide is endogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is native to that first species. In an embodiment, the amino acid sequence of the recombinant polypeptide is the same as or is substantially the same as, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from, a polypeptide encoded by the endogenous genome of the cell. In an embodiment, the recombinant polypeptide and the cell are from different species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a non-human, e.g., a rodent, e.g., a CHO, or an insect cell. In an embodiment, the recombinant polypeptide is exogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In an embodiment, the

recombinant polypeptide is a human polypeptide or protein which does not differ in amino acid sequence from a naturally occurring isoform of the human polypeptide or protein. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide or protein at no more than 1 , 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of its amino acid residues.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

MODULATION OF THE PROTEIN DEGRADATION PATHWAY

During cell line construction, e.g., recombinant cell line construction, cellular processes are triggered within the cell when the folding and modification capabilities of the cell are exceeded. The unfolded protein response (UPR) during ER stress and ER-associated degradation (ERAD) are key cellular processes that deal with misfolded or unassembled proteins during protein synthesis. Key responses that are activated by these processes include ER chaperone up-regulation, reduction in global protein synthesis by reducing translation initiation and, ultimately, if the stress persists, apoptosis (Schroder & Kaufman 2005; Chakrabarti et al. 201 1). It is

proposed that 30% of newly translated polypeptides are targeted for degradation, possibly as a result of misfolding (Du et al. 2013; Schubert et al. 2000; Yewdell & Nicchitta 2006). It is thought that these processes not only help to maintain protein quality but also provide an important source of 'recycled' amino acids as new building blocks for new polypeptide synthesis. It is therefore important that the cell has mechanisms in place to deal with, and recycle, the components of incorrectly folded polypeptides and maintains a quality control process.

ERAD requires that polypeptides/proteins in the ER destined for destruction are transported back out of the ER to the cytosol, e.g., retrotranslocated, where they are degraded by the proteasome (Olzmann et al. 2013). If homeostasis cannot be restored then the processes activated by the UPR can ultimately lead to apoptosis. The UPR provides the cell with the capability to adjust the ER capacity during periods of high demand and an element of UPR induction is thought to be beneficial to recombinant protein yield, however excessive and long term activation can be detrimental to the cell and culture viability.

The present disclosure is based, in part, on the results of a study to determine whether the capability of the cells to initiate protein turnover from the ER is linked to the ability of cells to produce a recombinant polypeptide, and further, whether inhibition of the protein degradation pathway, e.g., by inhibiting proteasome activity or polypeptide transport out of the ER during ERAD, could be used to select for cells with enhanced capability of these processes that would in turn reflect their ability to produce recombinant protein. The present disclosure provides methods and compositions for using inhibitors of protein degradation for identifying or selecting cells that have the capacity for high production of a product, e.g., a recombinant polypeptide, and for generating recombinant cell lines that have increased production, e.g., produce higher yields of a recombinant polypeptide product, and/or can produce higher quality recombinant polypeptide products.

Inhibitors of Protein Degradation

The methods described herein include contacting a cell, or a population of cells, with an inhibitor of protein degradation. In an embodiment, the inhibitors of protein degradation described herein inhibits or reduces the activity of a process or a protein involved in the protein degradation pathway, e.g., proteasome degradation, the ER-associated degradation pathway,

and/or the ubiquitin pathway. As described herein, the inhibitor of protein degradation can include, but is not limited to, a proteasome inhibitor, an ubiquitin pathway inhibitor, or an ERAD inhibitor. In an embodiment, the inhibitor may be reversible or irreversible. In embodiments, the inhibitor can be a small molecule, a nucleic acid, or a polypeptide.

The proteasome is a large, multienzyme complex comprising one or more protein-digesting enzymes, e.g., proteases. The 26S proteasome comprises a 20S proteolytic core subunit and one or two 19S regulatory particles. The 20S core contains three types of active sites, e.g. proteolytic or peptidase sites. In embodiments, the proteasome inhibitors described herein can inhibit or reduce the activity of any component of the proteasome. In an embodiment, the proteasome inhibitor binds to and/or inhibits one or more active sites of a component of the proteasome, e.g., one or more of the active sites of the 20S proteolytic core. In an embodiment, the proteasome inhibitor inhibits or reduces the activity of the 20S proteolytic core. In an embodiment, the proteasome inhibitor inhibits or reduces the activity of the 19S regulatory particle. In some embodiments, the proteasome inhibitor inhibits or reduces the activity of a protease, e.g., a trypsin protease, a chymotrypsin protease, or a calpain. In one embodiment, the proteasome inhibitor reduces the activity of the proteasome or of a component of the proteasome by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the activity of a proteasome or a component thereof in the absence of the inhibitor.

In an embodiment, the proteasome inhibitor is selected from MG132 or epoximicin. Other exemplary proteasome inhibitors are known in the art, and include, but are not limited to Z-Leu-Leu-Leu-B(OH)2 (MG262), bortezomib (PS-341 , Velcade, pyrazylcarbonyl-Phe-Leu-boronate), carfilzomib (PR-171), marizomib, MLN9708 (Ninlaro), ixazomib (MLN2238), oprozomib (ONX 0912), delanzomib (CEP-18770), CEP1612, celastrol, ONX-0914 (PR-957), disulfiram, epigallocatchin-3-gallate, salinosporamide A, AMI 14, lactacystin, ps-341 , eponomicin, Z-Leu-Leu-LeuVS, MG1 15, calpeptin, PSI, and calpain inhibitor II. Other exemplary proteasome inhibitors include NVLS, PS-519, antiprotealide, fluorosalinosporamide, and cinnabaramides (e.g., Cinnabaramides A~G).

The proteasome inhibitor may be any one described in Dick, et al., Biochem. 30: 2725 (1991); Goldberg, et al, Nature 357: 375 (1992); Goldberg, Eur. Biochem. 203: 9 (1992);

Orlowski, Biochem. 29: 10289 (1989); Rivett, et al, Archs. Biochem. Biophys. 218: 1 (1989);

Rivett, et al., J. Biol. Chem. 264: 12, 215 (1989); Tanaka, et al, New Biol. 4: 1 (1992), and U.S. Pat. No. 5,693,617.

The proteasome inhibitor can be a peptide aldehyde or a peptide alpha-keto esters containing a hydrophobic residue in the PI position as described in Vinitsky, et al. (Biochem. 31 : 9421 (1992), see also Orlowski, et al., Biochem. 32: 1563 (1993)), which were characterized as inhibitors of the chymotrypsin-like activity of the proteasome. The proteasome inhibitor can be a tripeptide, e.g., Ac-Leu-Leu-Leu-H, Ac-Leu-Leu-Met-OR, Ac-Leu-Leu-Nle-OR, Ac-Leu-Leu-Leu-OR, Ac-Leu-Leu-Arg-H, Z-Leu-Leu-Leu-H, Z-Arg-Leu-Phe-H and Z-Arg-Ile-Phe-H, where OR, along with the carbonyl of the preceding amino acid residue, represents an ester group.

The proteasome inhibitor can be an inhibitor of a chymotrypsin-like protease as disclosed by Siman, et al. (WO 01/13904). These inhibitors have the formula R-A4-A3-A2-Y, wherein R is hydrogen, or an N-terminal blocking group; A4 is a covalent bond, an amino acid or a peptide; A3 is a covalent bond, a D-amino acid, Phe, Tyr, Val or a conservative amino acid substitution of Val; A2 is a hydrophobic amino acid or lysine or a conservative amino acid substitution thereof, or when A4 includes at least two amino acids, A2 is any amino acid; and Y is a group reactive with the active site of said protease. The proteasome inhibitor can also be a peptide ketoamide, ketoacid, or ketoester useful in inhibiting serine proteases and cysteine proteases, as described in Powers (WO 92/12140). The proteasome inhibitor can also be a calpain inhibitor compound as described in Bartus, et al. (WO 92/1850).

Other proteasome inhibitors include, but are not limited to: Calpain Inhibitor I, MG101 , Calpain Inhibitor II, Fraction I (Frl, Hela), Fraction II (FII), clasto-Lactacystin beta-lactone (omuralide), Lactacystin, MG-115, Antiserum to NEDD8, PA28 Activator, 20S Proteasome, Polyclonal Antibody to Proteasome 20S alpha-Type 1 Subunit, Polyclonal Antibody to

Proteasome 26S Subunit SI 0B, Polyclonal Antibody to Proteasome 26S Subunit S2, Polyclonal Antibody to Proteasome 26S Subunit S4, Polyclonal Antibody to Proteasome 26S Subunit S5A, Polyclonal Antibody to Proteasome 26S Subunit S6, Polyclonal Antibody to Proteasome 26S Subunit S6', Polyclonal Antibody to Proteasome 26S Subunit S7, Polyclonal antibody to Proteasome 26S Subunit S8, Polyclonal antibody to Proteasome Activator PA28 Alpha, polyclonal antibody to Proteasome Activator PA28 Gamma, Polyclonal antibody to Proteasome Activator PA700 Subunit 10B, 26S Proteasome Fraction, Proteasome Inhibitor I, Proteasome Inhibitor II, Proteasome Substrate I (Fluorogenic), Proteasome Substrate II (Fluorogenic),

Proteasome Substrate III (Fluorogenic), Proteasome Substrate TV (Fluorogenic), S-100 Fraction, SUMO-l/Sentrin-1 (1-101), SUMO-l/Sentrin-1 (1-97), Antiserum to SUMO-l/Sentrin-1 , UbclO, Ubc5b, Ubc5c, Ubc6, Ubc7, Antiserum to Ubc9, Ubc9, UbCH2/E2-14K, UbCH3/Cdc34, UbCH5a, Ubiquitin Activating Enzyme (El), Ubiquitin Activating Enzyme (El), Ubiquitin Aldehyde, Ubiquitin Conjugating Enzyme Fractions, Ubiquitin C-terminal Hydrolase, Ubiquitin K48R, Methylated Ubiquitin, GST-Ubiquitin, (His)6 Ubiquitin, Ubiquitin-AMC, Ubiquitin-Sepharose.

In addition to known proteasome inhibitors, a proteasome inhibitor useful in the methods described herein can be routinely tested for their ability to inhibit proteasome activity. Various strategies for the identification of such inhibitors are exemplified in the art. For example, small molecule libraries, often comprising extracts from plants or more simple organisms, may be screened for their ability to inhibit the proteasome or specific protease types. Alternatively, a rational design approach may be applied using, for example, peptide or peptidomimetic compounds designed specifically to interact with the active site of a proteasome component (see e.g., Siman, et al., WO91/13904; Powers, et al., in Proteinase Inhibitors, Barrett, et al. (eds.), Elsevier, pp. 55-152 (1986)). The inhibitors can be stable analogs of catalytic transition states such as Z-Gly-Gly-Leu-H, which inhibits the chymotrypsin-like activity of the proteasome (Orlowski, Biochemistry 29: 10289 (1990); see also Kennedy and Schultz, Biochem. 18: 349 (1979)).

147 (4) 1360 (1991); Odcc, et al, Bhiochem. 30 (8): 2217 (1991); Vijayalakshmi, et al, Bhiochem. 30 (8) 2175 (1991); Work, et al., Thrombosis and Haemostasis 64 (1): 133 (1990);

Powers, et al., J. Cell. Biochem. 39 (1): 33 (1989); Powers, et al., Proteinase Inhibitors, Barrett et al., Eds., Elsevier, pp. 55-152 (1986); Powers et al, Biochem 29 (12): 3108 (1990); Oweida, et al, thrombosis Res. 58 (2): 391 (1990); Hudig, et al, Mol. Immunol. 26 (8): 793 (1989);

Orlowski, et al., Arch. Biochem. and Biophys. 269 ​​(1): 125 (1989); Zunino, et al., Biochem. and Biophys. Acta 967 (3): 331 (1988); Kam, et al, Biochem. 21 (1): 2547 (1988); Parkes, et al, Biochem. J. 230: 509 (1985); Green, et al, J. Biol. Chem. 256: 1923 (1981); Angliker, et al, Biochem. J. 241: 871 (1987); Puri, et al, Arch. Biochem. Biophys. 27: 346 (1989); Hanada, et al, Proteinase Inhibitors: Medical and Biological Aspects, Katunuma, et al, Eds, Springer-Verlag pp... 25-36 (1983); Kajiwara et al, Biochem. Int. 15: 935 (1987); Rao et al, Thromb. Res. 47: 635 (1987); Tsujinaka, et al., Biochem. Biophys. Res. Common. 153: 1201 (1988)).

Proteasome activity can be measured by proteasome activity assays known in the art and as described herein, e.g., in Examples 1 and 4.

Ubiquitination plays a critical role in protein degradation. Ubiquitin is a small, 8.5 kDa, protein that is attached to a substrate protein via an isopeptide bond between the carboxylic acid group of the ubiquitin' s glycine and the epsilon amino group of a lysine in the substrate protein. Ubiquitination of a substrate protein occurs in three main steps: 1) activation of the ubiquitin by ATP-dependent El ubiquitin activating enzyme (also referred to herein as El); 2) transfer of the ubiquitin from the E 1 to the active site of an E2 ubiquitin conjugating enzyme (also referred to herein as E2); and 3) ligation of the ubiquitin to the substrate protein via an E3 ubiquitin ligase (also referred to herein as E3), e.g., formation of the isopeptide bond between the ubiquitin molecule and the substrate protein. Ubiquitinated proteins are targeted for degradation by the proteasome. In one embodiment, the ubiquitin pathway inhibitor reduces the activity of the one or more components of the ubiquitin pathway by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, as compared to the activity of one or more components of the ubiquitin pathway in the absence of the ubiquitin pathway inhibitor.

In an embodiment, the ubiquitin pathway inhibitors described herein can inhibit any of the steps or enzymes involved in the ubiquitination cascade. For example, an ubiquitination inhibitor can inhibit or reduce the activity of one or more of, e.g., one, two, or all of, El, E2, or E3. In an embodiment, the ubiquitin pathway inhibitor inhibits or reduces the activity of any El , E2, or E3 known in the art. For example, E3 ubiquitin ligases include, but are not limited to,

APC/C ubiquitin ligase, HECT E3 ubiquitin ligase, β-TrCPl ligase, MDM2, and the SCF ubiquitin ligase.

In an embodiment, the ubiquitin pathway inhibitor is (-)-parthenolide, thalidomide, TAME, AOl (Tocris, Cat. No. 5397), Apcin (R&D Systems, Cat. No. I-444),GS143 (Tocris, Cat No. 5636), Heclin (Tocris, Cat. No. 5433), HLI373 (Tocris, Cat. No. 3503), NSC 6681 1 (Tocris, Cat. No. 2936), NSC-687852 (Biovision, Cat. No. 2021-5), NAB 2 (R&D Systems, Cat. No. 5131), Nutlin-3 (Tocris, Cat. No. 3984), PRT 4165 (Tocris, Cat. No. 5047), PTC 209 (Tocris, Cat. No. 5191), RITA (Tocris, Cat. No. 2443), SKPin CI (Tocris, Cat. No. 4817), SMER3 (Tocris, Cat. No. 437), SP141 (Tocris, Cat. No. 5332), SZL Pl -31 (Tocris, Cat. No. 5076), TAME hydrochloride (Tocris, Cat. No. 4506), proTAME (R&D Systems, Cat No. 1-440), or TCID (Biovision, Cat. No. 2204-5).

Ubiquitination can be measured by ubiquitination assays known in the art.

Elimination of misfolded proteins from the ER by ER-associated degradation involves, for example, identification and tagging of misfolded proteins to be degraded, retro-translocation of the misfolded protein from the ER lumen into the cytosol, ubiquitination of the protein, and delivery to the proteasome for degradation. A protein complex, p97-Ufdl-Npl4 ATPase complex hydro lyzes ATP to dislocate ubiquitinated proteins into the cytosol. A p97-associated deubiquitinating enzyme prevents the components of the p97-Ufdl-Npl4 ATPase complex itself from being targeted, e.g., ubiquitinated, and degraded by the proteasome. Thus, in an embodiment, an ERAD inhibitor inhibits or reduces the activity of one or more of the components of the p97-Ufdl -Npl4 ATPase complex, e.g., inhibits or reduces the activity of the ATPase of the p97-Ufdl-Npl4 ATPase complex. In one embodiment, the ERAD inhibitor reduces the activity of the one or more components of the ERAD pathway by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, as compared to the activity of one or more components of the ERAD pathway in the absence of the ERAD inhibitor.

In one embodiment, an ERAD inhibitor inhibits or reduces the activity of one or more components of the ERAD pathway. Components of the ERAD pathway include, but are not limited to, Hsp70 family members, Hsp40 family members, Hsp90 family members, nucleotide exchange factors (NEFs) (e.g., Nhs, Bag-1), Rotl , calnexin, alreticulin, a-mannosidase (Mnsl), ER Degradation Enhancing a-mannosidase -like protein (EDEM), Kar2, Sec61 complex

(components include, e.g., Sec61a and Sec61y), protein disulfide isomerases (PDI), Yos9, Cdc48p, p97, VCP/p97 complex, Hrdl, DoalO, Ubcl, Cuel , Ubc6, and Ubc7.

In an embodiment, an ERAD inhibitor includes, but is not limited to, eeyarestatin I (also referred to as ESI), cotransin, CAM741 , apratoxin A, exotoxin A, HU -7293, decatransin, valinomycin, mycolactone, NSC 630668-R/l (also referred to as R/l), MAL3-39, MAL3-101, E6 Berbamine, Ophiobolin A, CADA cyclotriazadisulfoniamide, e.g., inhibitors described in Kalies et al., 2015, Traffic, 16: 1027-1038; incorporated herein by reference.

In one embodiment, one or more inhibitors of protein degradation, as described herein, are used in the methods described herein. For example, two, three, four, or five, or more, inhibitors of protein degradation as described herein are used. In an embodiment, the two or more inhibitors of protein degradation may disrupt the same process in the protein degradation pathway, e.g., the two or more inhibitors inhibit or reduce proteasome activity. In an alternative embodiment, the two or more inhibitors of protein degradation disrupt different processes in the protein degradation pathway, e.g., one inhibitor inhibits or reduces proteasome activity while another inhibitor inhibits or reduces ERAD activity or the ubiquitin pathway.

Determination of the appropriate concentrations of the inhibitors of protein degradation is well within the skill of the ordinary skilled artisan. Assay for determining concentrations of such inhibitors, e.g., determining appropriate concentrations of the inhibitor for desired culture viability, and/or protein degradation inhibition, e.g., proteasome inhibition, are described herein and in the Examples herein. The appropriate concentration of the inhibitors of protein degradation for use in the methods described herein may differ in different cell types or cells from different species. In the embodiments described herein, the inhibition or reduction of proteasome activity is determined as compared to a cell that has not been contacted with the proteasome inhibitor, or as compared to a reference cell that has been contacted with the proteasome inhibitor.

In some embodiments, a low concentration of the inhibitor of protein degradation is used in the methods described herein. Low concentrations of the inhibitor of protein degradation may be preferred for cells that have undergone one or more selection steps prior to contacting with, e.g., exposure to, the inhibitor of protein degradation as described herein. By way of example, a low concentration of the inhibitor of protein degradation is administered to the cell after selection is performed to identify cells that have stably integrated an exogenous nucleic acid into the genome.

In one embodiment, the concentration of the inhibitor of protein degradation is a concentration that results in the inhibition or reduction of protein degradation, e.g., inhibition or reduction of activity of one or more component of the proteasome, the ubiquitin pathway, or the ERAD pathway as described herein. In an embodiment, the concentration of the inhibitor of protein degradation is a concentration that results in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more culture viability (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of cells are viable or survive in a population of cells contacted by, e.g., cultured in the presence of, an inhibitor of protein degradation described herein) in a culture or population of cells that are contacted by, e.g., cultured in the presence of, the inhibitor of protein degradation. In an embodiment, the concentration of the inhibitor of protein degradation is a concentration at which some portion of the cells contacted by the inhibitor of protein degradation continue to proliferate, e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of cells continue to proliferate when contacted by, e.g., cultured in the presence of, the inhibitor of protein degradation. In some embodiments, the concentration of an inhibitor of protein degradation described herein results in less than 20%, e.g., 15%, 10%, 5%, 1%, 0.5%, or 0.1%, of the total number of cells remaining after contacting the cell with, e.g., culturing the cell in the presence of, the inhibitor of protein degradation.

In one embodiment, the inhibitor of protein degradation is MG-132. MG-132 has been used at 1.5 μΜ to induce apoptosis (Meriin et al. 1998). In an embodiment, the concentration of MG-132 suitable for the methods described herein is less than 1.5 μΜ, less than 1.25 μΜ, less than 1.0 μΜ, less than 0.9 μΜ, less than 0.8 μΜ, less than 0.7 μΜ, less than 0.6 μΜ, less than 0.5 μΜ, less than 0.4 μΜ, less than 0.3 μΜ, less than 0.2 μΜ, less than 0.1 μΜ, less than 0.09 μΜ, less than 0.08 μΜ, less than 0.07 μΜ, less than 0.06 μΜ, less than 0.05 μΜ, less than 0.04 μΜ, less than 0.03 μΜ, less than 0.02 μΜ, less than 0.01 μΜ, or less than 0.005 μΜ. In an embodiment, the concentration of MG-132 is about 0.1 μΜ or 0.0625 μΜ.

In one embodiment, the inhibitor of protein degradation is epoxomicin. Concentrations of epoxomicin at 0.04-0.08 μΜ has been shown to inhibit chymotrypsin activity of the proteasome (Meng et al. 1999). In an embodiment, the concentration of epoxomicin suitable for

the methods described herein is less than 0.08 μΜ, less than 0.07 μΜ, less than 0.06 μΜ, less than 0.05 μΜ, less than 0.04 μΜ, less than 0.03 μΜ, less than 0.02 μΜ, less than 0.01 μΜ, less than 0.009 μΜ, less than 0.008 μΜ, less than 0.007 μΜ, less than 0.006 μΜ, less than 0.005 μΜ, less than 0.004 μΜ, less than 0.003 μΜ, less than 0.002 μΜ, less than 0.001 μΜ, less than 0.009 μΜ, less than 0.008 μΜ, less than 0.007 μΜ, less than 0.006 μΜ, less than 0.005 μΜ, less than 0.004 μΜ, less than 0.003 μΜ, less than 0.002 μΜ, or less than 0.001 μΜ. In an embodiment, the concentration of epoxomicin is about 0.05 μΜ or 0.025 μΜ.

In one embodiment, the inhibitor of protein degradation is eeyarestatin I. The

concentration of 8 μΜ eeyarestatin I has previously been shown in vivo to be sufficient to block ER translocation (Cross et al. 2009). In an embodiment, the concentration of eeyarestatin I suitable for the methods described herein is less than 20 μΜ, less than 15 μΜ, less than 14 μΜ, less than 13 μΜ, less than 12 μΜ, less than 11 μΜ, less than 10 μΜ, less than 9 μΜ, less than 8 μΜ, less than 7 μΜ, less than 6 μΜ, less than 5 μΜ, less than 4 μΜ, less than 3 μΜ, less than 2 μΜ, less than 1 μΜ, or less than 0.5 μΜ.

In any of the methods described herein for evaluation or identification of cells with certain characteristics, e.g., susceptibility to the inhibitors of protein degradation, one or more cells can be screened at the same time in a high throughput format. For example, cells can be grown in separate aliquots, e.g., in different wells of a multi-well plate, where each individual aliquot of cells is cultured or selected under different conditions. The different conditions can include different concentrations of the inhibitor of protein degradation, additional selection steps, or introduction of different exogenous nucleic acids. In such embodiments, comparison of a value of one or more parameter related to cell function determined from each aliquot can be used to identify, select, or classify a cell as a high producer, or as a cell for further development as a high production cell line.

PRODUCTS AND NUCLEIC ACIDS ENCODING THEM

Provided herein are methods for identifying, selecting, or making a cell or cell line capable of producing high yields of a product. The products encompassed by the present disclosure include, but are not limited to, molecules, nucleic acids, polypeptides (e.g., recombinant polypeptides), or hybrids thereof, that can be produced by, e.g., expressed in, a cell.

In some embodiments, the cells are engineered or modified to produce the product. Such modifications include the introducing molecules that control or result in production of the product. For example, a cell is modified by introducing an exogenous nucleic acid that encodes a polypeptide, e.g., a recombinant polypeptide, and the cell is cultured under conditions suitable for production, e.g., expression and secretion, of the polypeptide, e.g., recombinant polypeptide. In another example, a cell is modified by introducing an exogenous nucleic acid that controls, e.g., increases, expression of a polypeptide that is endogenously expressed by the cell, such that the cell produces a higher level or quantity of the polypeptide than the level or quantity that is endogenously produced, e.g., in an unmodified cell.

In embodiments, the cell or cell line identified, selected, or generated by the methods described herein produces a product, e.g., a recombinant polypeptide, useful in the treatment of a medical condition, disorder or disease. Examples of medical conditions, disorders or diseases include, but are not limited to, metabolic disease or disorders (e.g., metabolic enzyme deficiencies), endocrine disorders (e.g., hormone deficiencies), haemostasis, thrombosis, hematopoietic disorders, pulmonary disorders, gastro -intestinal disorders, immunoregulation (e.g., immunodeficiency), infertility, transplantation, cancer, and infectious diseases.

In some embodiments, the product is an exogenous protein, e.g., a protein that is not naturally expressed by the cell. The product can be a therapeutic protein or a diagnostic protein, e.g., useful for drug screening. The therapeutic or diagnostic protein can be an antibody molecule, e.g., an antibody or an antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, or a structural and/or functional fragment or hybrid of any of these.

In one embodiment, the product, e.g., recombinant polypeptide, is an antibody molecule. Products encompassed herein comprise diagnostic and therapeutic antibody molecules. A diagnostic antibody molecule includes an antibody, e.g., a monoclonal antibody or antibody fragment thereof, useful for imaging techniques. A therapeutic antibody molecule is suitable for administration to subjects, e.g., for treatment or prevention of a disease or disorder.

An antibody molecule is a protein, or polypeptide sequence derived from an

immunoglobulin molecule which specifically binds with an antigen. In an embodiment, the antibody molecule is a full-length antibody or an antibody fragment. Antibodies and multiformat proteins can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. In an embodiment, the antibody is a monoclonal antibody. The antibody may be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, or IgE antibody. In one embodiment, the antibody is an IgGl , IgG2, IgG3, or IgG4 antibody.

"Antibody fragment" refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1 126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a flbronectin type III (Fn3) (see U.S. Patent No.:

6,703,199, which describes flbronectin polypeptide minibodies).

Exemplary products, e.g., polypeptides, e.g., recombinant polypeptides, produced in the methods or cells described herein are provided in the tables below.

Table 1. Exemplary Products

hormone (GHRH) ChiRhoStim (human peptide), SecreFlo (porcine

Secretin peptide)

Thyroid stimulating hormone Thyrogen

(TSH), thyrotropin

Blood Factor Vila Novo Seven

Clotting/Coagulation Factor VIII Bioclate, Helixate, Kogenate, Recombinate, ReFacto

Factors

Factor IX Benefix

Antithrombin III (AT-III) Thrombate III

Protein C concentrate Ceprotin

Cytokine/ Growth Type I alpha-interferon Infergen

factor Interferon- an3 (IFNan3) Alferon N

Interferon-pia (rIFN- β) Avonex, Rebif

Interferon-pib (rIFN- β) Betaseron

Interferon-ylb (IFN γ) Actimmune

Aldesleukin (interleukin 2(IL2), Proleukin

epidermal theymocyte activating

factor; ETAF

Palifermin (keratinocyte growth

Kepivance

factor; GF)

Regranex

Becaplemin (platelet-derived

growth factor; PDGF)

Anakinra (recombinant IL1 Anril, Kineret

antagonist)

Antibody molecules Bevacizumab (VEGFA mAb) Avastin

Cetuximab (EGFR mAb) Erbitux

Panitumumab (EGFR mAb) Vectibix

Alemtuzumab (CD52 mAb) Campath

Rituximab (CD20 chimeric Ab) Rituxan

Trastuzumab (HER2/Neu mAb) Herceptin

Abatacept (CTLA Ab/Fc fusion) Orencia

Adalimumab (TNFa mAb) Humira

Etanercept (TNF receptor/Fc Enbrel

fusion)

Infliximab (TNFa chimeric mAb) Remicade

Alefacept (CD2 fusion protein) Amevive

Efalizumab (CD1 la mAb) Raptiva

Natalizumab (integrin a4 subunit Tysabri

mAb)

Soliris

Eculizumab (C5mAb)

Orthoclone, OKT3

Muromonab-CD3

Other: Insulin Humulin, Novolin

Fusion Hepatitis B surface antigen Engerix, Recombivax HB

proteins/Protein (HBsAg)

vaccines/Peptides HPV vaccine Gardasil

OspA LYMErix

Anti-Rhesus(Rh) immunoglobulin Rhophylac

G

Fuzeon

Enfuvirtide

Spider silk, e.g., fibrion

QMONOS

In another embodiment, the product is a bispecific molecule. Bispecific molecules, as described herein, include molecules that can bind to two or more distinct antigens or targets. In an embodiment, a bispecific molecule comprises antibody fragments. In one embodiment, the bispecific molecule comprises a bispecific antibody, a bispecific antibody fusion protein, or a bispecific antibody conjugate, a Bi-specific T cell Engager (BiTE) molecule, a Dual Affinity Re-Targeting (DART) Molecule, a Dual Action Fab (DAF) molecule, a nanobody, or other arrangement of antibody fragments resulting in a molecule having the ability to recognize or bind to two distinct antigens.

Table 2. Exemplary Products, e.g., Bispecific Molecules

University of Minnesota) to diphtheria toxin to tumor or lymphoma

(Chugai, Roche) factor X

Other exemplary therapeutic or diagnostic proteins include, but are not limited to any protein described in Tables 1 -10 of Leader et al., "Protein therapeutics: a summary and pharmacological classification", Nature Reviews Drug Discovery, 2008, 7:21 -39 (incorporated herein by reference); or any conjugate, variant, analog, or functional fragment of the recombinant polypeptides described herein.

Other recombinant products include non-antibody scaffolds or alternative protein scaffolds, such as, but not limited to: DARPins, affibodies and adnectins. Such non-antibody scaffolds or alternative protein scaffolds can be engineered to recognize or bind to one or two, or more, e.g., 1, 2, 3, 4, or 5 or more, different targets or antigens.

Also provided herein are nucleic acids, e.g., exogenous nucleic acids that encode the products, e.g., polypeptides, e.g., recombinant polypeptides described herein. The nucleic acid sequences coding for the desired recombinant polypeptides can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the desired nucleic acid sequence, e.g., gene, by deriving the nucleic acid sequence from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the nucleic acid encoding the recombinant polypeptide can be produced synthetically, rather than cloned. Recombinant DNA techniques and technology are highly advanced and well established in the art. Accordingly, the ordinarily skilled artisan having the knowledge of the amino acid sequence of a recombinant polypeptide described herein can readily envision or generate the nucleic acid sequence that would encode the recombinant polypeptide.

In some embodiments, the exogenous nucleic acid controls the expression of a product that is endogenously expressed by the host cell. In such embodiments, the exogenous nucleic acid comprises one or more nucleic acid sequences that increase the expression of the endogenous product (also referred to herein as "endogenous product transactivation sequence"). For example, the nucleic acid sequence that increases the expression of an endogenous product comprises a constitutively active promoter or a promoter that is stronger, e.g., increases transcription at the desired site, e.g., increases expression of the desired endogenous gene product. After introduction of the exogenous nucleic acid comprising the endogenous product transactivation sequence, said exogenous nucleic acid is integrated into the chromosomal genome of the cell, e.g., at a preselected location proximal to the genomic sequence encoding the endogenous product, such that the endogenous product transactivation sequence increases the transactivation or expression of the desired endogenous product. Other methods for modifying a cell, e.g., introducing an exogenous nucleic acid, for increasing expression of an endogenous product is described, e.g., in U.S. Patent No. 5,272,071; hereby incorporated by reference in its entirety.

The expression of a product described herein is typically achieved by operably linking a nucleic acid encoding the recombinant polypeptide or portions thereof to a promoter, and

incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes or prokaryotes. Typical cloning vectors contain other regulatory elements, such as transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The nucleic acid sequences described herein encoding a product, e.g., a recombinant polypeptide, or comprising a nucleic acid sequence that can control the expression of an endogenous product, can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In embodiments, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Vectors derived from viruses are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.

A vector may also include, e.g., a signal sequence to facilitate secretion, a

polyadenylation signal and transcription terminator (e.g., from Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g. SV40 origin and ColEl or others known in the art) and/or elements to allow selection, e.g., a selection marker or a reporter gene.

In one embodiment, the vector comprising a nucleic acid sequence encoding a

polypeptide, e.g., a LMM or a recombinant polypeptide, further comprises a promoter sequence responsible for the recruitment of polymerase to enable transcription initiation for expression of the polypeptide, e.g., the LMM or recombinant polypeptide. In one embodiment, promoter sequences suitable for the methods described herein are usually associated with enhancers to drive high amounts of transcription and hence deliver large copies of the target

exogenous mRNA. In an embodiment, the promoter comprises cytomegalovirus (CMV) major immediate early promoters (Xia, Bringmann et al. 2006) and the S V40 promoter (Chernajovsky, Mory et al. 1984), both derived from their namesake viruses or promoters derived therefrom. Several other less common viral promoters have been successfully employed to drive transcription upon inclusion in an expression vector including Rous Sarcoma virus long terminal repeat (RSV-LTR) and Moloney murine leukaemia virus (MoMLV) LTR (Papadakis, Nicklin et al. 2004). In another embodiment, specific endogenous mammalian promoters can be utilized to drive constitutive transcription of a gene of interest (Pontiller, Gross et al. 2008). The CHO specific Chinese Hamster elongation factor 1 -alpha (CHEF 1 a) promoter has provided a high yielding alternative to viral based sequences (Deer, Allison 2004). In addition to promoters, the vectors described herein further comprise an enhancer region as described above; a specific nucleotide motif region, proximal to the core promoter, which can recruit transcription factors to upregulate the rate of transcription (Riethoven 2010). Similar to promoter sequences, these regions are often derived from viruses and are encompassed within the promoter sequence such as hCMV and SV40 enhancer sequences, or may be additionally included such as adenovirus derived sequences (Gaillet, Gilbert et al. 2007).

In one embodiment, the vector comprising a nucleic acid sequence encoding a product, e.g., a polypeptide, e.g, a recombinant polypeptide, described herein further comprises a nucleic acid sequence that encodes a selection marker. In one embodiment, the selectable marker comprises glutamine synthetase (GS); dihydrofolate reductase (DHFR) e.g., an enzyme which confers resistance to methotrexate (MTX); or an antibiotic marker, e.g., an enzyme that confers resistance to an antibiotic such as: hygromycin, neomycin (G418), zeocin, puromycin, or blasticidin. In another embodiment, the selection marker comprises or is compatible with the Selexis selection system (e.g., SUREtechnology Platform™ and Selexis Genetic Elements™, commercially available from Selexis SA) or the Catalant selection system.

In one embodiment, the vector comprising a nucleic acid sequence encoding a recombinant product described herein comprises a selection marker that is useful in identifying a cell or cells comprise the nucleic acid encoding a recombinant product described herein. In another embodiment, the selection marker is useful in identifying a cell or cells that comprise the integration of the nucleic acid sequence encoding the recombinant product into the genome, as described herein. The identification of a cell or cells that have integrated the nucleic acid sequence encoding the recombinant protein can be useful for the selection and engineering of a cell or cell line that stably expresses the product.

Suitable vectors for use are commercially available, and include vectors associated with the GS Expression System™, GS Xceed™ Gene Expression System, or Potelligent® CHOK1SV technology available from Lonza Biologies, Inc, e.g., vectors as described in Fan et al., Pharm. Bioprocess. (2013); l(5):487-502, which is incorporated herein by reference in its entirety. GS expression vectors comprise the GS gene, or a functional fragment thereof (e.g., a GS mini-gene), and one or more, e.g., 1 , 2, or 3, or more, highly efficient transcription cassettes for expression of the gene of interest, e.g., a nucleic acid encoding a recombinant polypeptide described herein. A GS mini-gene comprises, e.g., consists of, intron 6 of the genomic CHO GS gene. In one embodiment, a GS vector comprises a GS gene operably linked to a SV40L promoter and one or two polyA signals. In another embodiment, a GS vector comprises a GS gene operably linked to a SV40E promoter, SV40 splicing andpolyadenylation signals. In such embodiments, the transcription cassette, e.g., for expression of the gene of interest or recombinant polypeptide described herein, includes the hCMV-MIE promoter and 5' untranslated sequences from the hCMV-MIE gene including the first intron. Other vectors can be constructed based on GS expression vectors, e.g., wherein other selection markers are substituted for the GS gene in the expression vectors described herein.

Vectors suitable for use in the methods described herein include, but are not limited to, other commercially available vectors, such as, pcDNA3.1/Zeo, pcDNA3.1/CAT,

pcDNA3.3TOPO (Thermo Fisher, previously Invitrogen); pTarget, HaloTag (Promega); pUC57 (GenScript); pFLAG-CMV (Sigma-Aldrich); pCMV6 (Origene); pEE12 or pEE14 (Lonza Biologies), or pBK-CMV/ pCMV-3Tag-7/ pCMV-Tag2B (Stratagene).

CELLS AND CELL CULTURE

In one aspect, the present disclosure relates to methods for evaluating, classifying, identifying, selecting, or making a cell or cell line that produces a product, e.g., a recombinant polypeptide as described herein. In another aspect, the present disclosure relates to methods and compositions for evaluating, classifying, identifying, selecting, or making a cell or cell line with improved, e.g., increased, productivity and product quality.

In embodiments, the cell is a mammalian cell. In other embodiments, the cell is a cell other than a mammalian cell. In an embodiment, the cell is from mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, or cat. In embodiments, the cell is a mammalian cell, e.g., a human cell or a rodent cell, e.g., a hamster cell, a mouse cell, or a rat cell. In another embodiment, the cell is from a duck, parrot, fish, insect, plant, fungus, or yeast. In one embodiment, the cell is an Archaebacteria. In an embodiment, the cell is a species of

Actinobacteria, e.g., Mycobacterium tuberculosis .

In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-out cell, a CHO FUT8 GS 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.). The CHO FUT8 knockout cell is, for example, the Potelligent® CHOK1 SV (Lonza Biologies, Inc.).

In another embodiment, the cell is a Hela, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cell), VERO, SP2/0, NSO, YB2/0, Y0, EB66, C127, L cell, COS, e.g., COS1 and COS7, QCl-3, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1 SV GS-KO, CHOS, CHO DG44, CHO DXB11 , and CHOZN, or any cells derived therefrom. In one embodiment, the cell is a stem cell. In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

In an embodiment, the cell is any one of the cells described herein that comprises an exogenous nucleic acid encoding a recombinant polypeptide, e.g., expresses a recombinant polypeptide, e.g., a recombinant polypeptide selected from Table 1 or 2.

In an embodiment, the cell culture is carried out as a batch culture, fed-batch culture, draw and fill culture, or a continuous culture. In an embodiment, the cell culture is a suspension culture. In one embodiment, the cell or cell culture is placed in vivo for expression of the recombinant polypeptide, e.g., placed in a model organism or a human subject.

In one embodiment, the culture media is free of serum. Serum-free and protein-free media are commercially available, e.g., Lonza Biologies.

Suitable media and culture methods for mammalian cell lines are well-known in the art, as described in U.S. Pat. No. 5,633,162 for instance. Examples of standard cell culture media for

laboratory flask or low density cell culture and being adapted to the needs of particular cell types are for instance: Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, lp. 173 ff, 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc.53, p288 ff. 1965) or Iscoves' modified DMEM lacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med. 1, p. 923 ff, 1978). For instance, Ham's F10 or F12 media were specially designed for CHO cell culture. Other media specially adapted to CHO cell culture are described in EP-481 791. It is known that such culture media can be supplemented with fetal bovine serum (FBS, also called fetal calf serum FCS), the latter providing a natural source of a plethora of hormones and growth factors. The cell culture of mammalian cells is nowadays a routine operation well-described in scientific textbooks and manuals, it is covered in detail e.g. in R. Ian Fresney, Culture of Animal cells, a manual, 4th edition, Wiley-Liss/N.Y., 2000.

Other suitable cultivation methods are known to the skilled artisan and may depend upon the recombinant polypeptide product and the host cell utilized. It is within the skill of an ordinarily skilled artisan to determine or optimize conditions suitable for the expression and production of the recombinant polypeptide to be expressed by the cell.

In one aspect, the cell or cell line comprises an exogenous nucleic acid that encodes a product, e.g., a recombinant polypeptide. In an embodiment, the cell or cell line expresses the product, e.g., a therapeutic or diagnostic product. Methods for genetically modifying or engineering a cell to express a desired polypeptide or protein are well known in the art, and include, for example, transfection, transduction (e.g., viral transduction), or electroporation.

Physical methods for introducing a nucleic acid, e.g., an exogenous nucleic acid or vector described herein, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY).

Chemical means for introducing a nucleic acid, e.g., an exogenous nucleic acid or vector described herein, into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water

emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

In embodiments, the integration of the exogenous nucleic acid into a nucleic acid of the host cell, e.g., the genome or chromosomal nucleic acid of the host cell is desired. Methods for determining whether integration of an exogenous nucleic acid into the genome of the host cell has occurred can include a GS/MSX selection method. The GS/MSX selection method uses complementation of a glutamine auxotrophy by a recombinant GS gene to select for high-level expression of proteins from cells. Briefly, the GS/MSX selection method comprises inclusion of a nucleic acid encoding glutamine synthetase on the vector comprising the exogenous nucleic acid encoding the recombinant polypeptide product. Administration of methionine sulfoximine (MSX) selects cells that have stably integrated into the genome the exogenous nucleic acid encoding both the recombinant polypeptide and GS. As GS can be endogenously expressed by some host cells, e.g., CHO cells, the concentration and duration of selection with MSX can be optimized to identify high producing cells with stable integration of the exogenous nucleic acid encoding the recombinant polypeptide product into the host genome. The GS selection and systems thereof is further described in Fan et al., Pharm. Bioprocess. (2013); l(5):487-502, which is incorporated herein by reference in its entirety.

Other methods for identifying and selecting cells that have stably integrated the exogenous nucleic acid into the host cell genome can include, but are not limited to, inclusion of a reporter gene on the exogenous nucleic acid and assessment of the presence of the reporter gene in the cell, and PCR analysis and detection of the exogenous nucleic acid.

In one embodiment, the cells selected, identified, or generated using the methods described herein are capable of producing higher yields of protein product than cells that are selected using only a selection method for the stable expression, e.g., integration of exogenous nucleic acid encoding the recombinant polypeptide. In an embodiment, the cells selected, identified, or generated using the methods described herein produce 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more of the product, e.g., recombinant polypeptide, as compared to cells that were not contacted with an inhibitor of protein degradation, or cells that were only selected for stable expression, e.g., integration, of the exogenous nucleic acid encoding the recombinant polypeptide.

EVALUATING, CLASSIFYING, SELECTING, OR IDENTIFYING A CELL

In one aspect, the disclosure features methods for evaluating a cell, e.g., a candidate cell, for capability of product production, e.g., recombinant polypeptide production. The results of such evaluation can provide information useful for selection or identification of cells for generating a cell or cell line that is a high production cell or cell line. In another embodiment, the responsive to the evaluation described herein, the cell or cell line can be classified, e.g., as a cell or cell line that has the capability of high production.

A high production cell or cell line is capable of producing higher yields of a recombinant polypeptide product than compared to a reference cell or a cell that has not been selected or generated by the methods described herein. In an embodiment, a high production cell line is capable of producing 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 g/L, 700 g/L, 800 g/L, 900 g/L,l g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L, 70 g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, or 100 g/L or more of a product, e.g., a recombinant polypeptide product. In an embodiment, a high production cell line is produces 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 g/L, 700 g/L, 800 g/L, 900 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L, 70 g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, or 100 g/L or more of a product, e.g., a recombinant polypeptide product. The quantity of product produced may vary depending on the cell type, e.g., species, and the recombinant polypeptide to be expressed. By way of example, a high production cell is capable of producing at least 1 g/L, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, or 25 g/L or more of a recombinant polypeptide, e.g., as described herein.

In embodiments where the product is difficult to express, the high production cell may produce lower concentrations of products, e.g., less than 1 g/L, however, the productivity is higher or increased than that observed for cells that have not been contacted with an inhibitor of protein degradation. For example, the level, amount, or quantity of the product produced by the identified or selected cell is increased, e.g., by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, as compared to the level,

amount, or quantity produced by a cell that has not been contacted by the inhibitor of protein degradation.

The methods described herein for evaluating a cell include evaluating the effect of an inhibitor of protein degradation on one or more parameters related to cell function. Parameters related to cell function include, but are not limited to, cell survival, culture viability, the ability to proliferate, the ability to produce a product, and protein degradation. In embodiments, the value of the effect of an inhibitor of protein degradation on one or more parameters related to cell function is compared to a reference value, for determining the effect of the inhibitor on the parameter related to cell function, e.g., for determining whether contacting the cell with the inhibitor results in an increase or decrease in one of the parameters related to cell function. In one embodiment, a cell can be selected or identified for development as a cell production line in response to the determination of an increases or decrease in one or more of the parameters related to cell function. In one embodiment, a cell can be identified as a high production cell, e.g., a cell capable of producing higher yields of a product, in response to the determination of an increase or decrease in one or more of the parameters related to cell function.

In any of the embodiments described herein, the reference value can be the value of the effect of the inhibitor of protein degradation on a parameter related to cell function of a reference cell, e.g., a cell with a predetermined productivity. Alternatively or in addition, in any of the embodiments described herein, the reference value can be the value of the parameter related to cell function of the same cell being tested, where the cell has not been contacted with the inhibitor, e.g., the value of the parameter was measured before contacting the cell with the inhibitor of protein degradation, or a separate aliquot of the cell that has not been contacted with the inhibitor of protein degradation.

In one embodiment, cell survival can be measured by determining or quantifying cell apoptosis, e.g., the number or amount of cells that have been killed or died in response to a concentration of an inhibitor of protein degradation described herein. An increase in cell survival comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, one-fold, two-fold, three-fold, four-fold, or five-fold or more increase in the number of cells, e.g., intact or live cells, remaining after contacting with the inhibitor of protein degradation. Alternatively, an increase in cell survival comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more decrease in the number of apoptotic cells after contacting with the inhibitor of

protein degradation. Methods for detecting cell survival or apoptosis are known in the art, e.g., Annexin V assays, and are described herein in the Examples.

In one embodiment, culture viability can be measured by determining or quantifying the number or amount of live cells, e.g., live cells in a culture or population of cell,, or cells that have a characteristic related to viability, e.g., proliferation markers, intact DNA, or do not display apoptotic markers. An increase in culture viability comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, one-fold, two-fold, three-fold, four-fold, or five-fold or more increase in the number of cells, e.g., intact or live cells, remaining after contacting with the inhibitor of protein degradation. Methods for determining culture viability are known in the art, and are described herein in Examples 1 and 3. Other methods for assessing culture viability include, but are not limited to, trypan blue exclusion methods followed by counting using a haemocytometer or Vi-CELL (Beckman-Coulter). Other methods for assessing culture viability can comprise determining viable biomass, and includes using radiofrequency impedance or capacitance (e.g., as described in Carvell and Dowd, 2006, Cytotechnology, 50:35-48), or using Raman spectroscopy (e.g., as described in Moretto et al., 2011 , American Pharmaceutical Review, Vol. 14).

In one embodiment, the ability of a cell to proliferate can be measured by quantifying or counting the number of cells, cell doublings, or growth rate of the cells. Alternatively, proliferating cells can be identified by analysis of the genomic content of the cells (e.g., replicating DNA), e.g., by flow cytometry analysis, or presence of proliferation markers, e.g., Ki67, phosphorylated cyclin-CDK complexes involved in cell cycle. An increase in the ability to proliferate comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, onefold, two-fold, three-fold, four-fold, or five-fold or more increase in the number of cells, or number of cells expressing a proliferation marker, after contacting the cell with the inhibitor of protein degradation. Alternatively, an increase in the ability to proliferate comprises a 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, one-fold, two-fold, three-fold, four-fold, or five-fold or more increase in the doubling or growth rate of the cells after contacting the cells with the inhibitor of protein degradation.

The methods provided herein are useful for identifying, selecting, or making a cell or cell line that has improved capacity for producing a recombinant polypeptide, e.g., a product. In one embodiment, the methods provided herein are also useful for identifying, selecting, or making a cell or cell line that produces an improved quality of the recombinant polypeptide.

In one embodiment, the ability of the cell to produce a product can be measured by determining or quantifying the amount or concentration of product that is produced. An increase in the ability to produce a product comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, one-fold, two-fold, three-fold, four-fold, or five-fold or more increase in protein production after contacting the cell with the inhibitor of protein degradation.

In one embodiment, the quality of the product, e.g., expressed recombinant polypeptide, can be measured by determining or quantifying the amount or concentration of properly folded product, functional product, or non-aggregated product. An increase in the quality of the product produced by the cell comprises a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, one-fold, two-fold, three-fold, four-fold, or five-fold or more increase in the amount or concentration of properly folded product, functional product, or non-aggregated product, e.g., expressed recombinant polypeptide, after contacting the cell with the inhibitor of protein degradation.

Methods of measuring increased protein production are well-known to those skilled in the art. For example, an increase in recombinant protein production might be determined at small-scale by measuring the titer in tissue culture medium by ELISA (Smales et al. 2004

Biotechnology Bioengineering 88:474-488). It can also be determined quantitatively by the ForteBio Octet, for example for high throughput determination of recombinant monoclonal antibody (mAb) concentration in medium (Mason et al. 2012 Biotechnology Progress 28:846-855) or at a larger-scale by protein A HPLC (Stansfield et al. 2007 Biotechnology

Bioengineering 97:410-424). Other methods for determining production of a product, e.g., a recombinant polypeptide described herein, can refer to specific production rate (qP) of the product, in particular the recombinant polypeptide in the cell and/or to a time integral of viable cell concentration (rVC). Recombinant polypeptide production or productivity, being defined as concentration of the polypeptide in the culture medium, is a function of these two parameters (qP and rVC), calculated according to Porter et al. (Porter et al. 2010 Biotechnology Progress 26:1446-1455). Methods for measuring protein production are also described in further detail in the Examples provided herein, e.g., Examples 1 and 5.

Methods for measuring improved quality of product produced by the cell lines generated as described herein are known in the art. In one embodiment, methods for determining the fidelity of the primary sequence of the expressed recombinant polypeptide product are known in the art, e.g., mass spectrometry. An increase in the amount or concentration of properly folded product, e.g., expressed recombinant polypeptide, can be determined by circular dichroism or assessing the intrinsic fluorescence of the expressed recombinant polypeptide. An increase in the amount or concentration of functional product can be tested using various functional assays depending on the identity of the recombinant polypeptide. For example, antibodies can be tested by the ELIS A or other immuno affinity assay.

METHODS FOR CELL LINE AND RECOMBINANT POLYPEPTIDE PRODUCTION

The current state of the art in both mammalian and microbial selection systems is to apply selective pressure at the level of the transcription of DNA into RNA. The gene of interest is tightly linked to the selection marker making a high level of expression of the selective marker likely to result in the high expression of the gene of interest. Cells which express the selection marker at high levels are able to survive and proliferate, those which do not are less likely to survive and proliferate, e.g., apoptose and/or die. In this way a population of cells can be enriched for cells expressing the selection marker and by implication the gene of interest at high levels. This method has proved very successful for expressing straightforward proteins.

However, for some proteins the bottleneck for expression is not the transcription of DNA into RNA but rather the correct folding and if necessary posttranslational modifications of the protein. Current selection systems targeting transcription do not apply pressure for the selection of cell lines that are good at producing and correctly processing proteins. In fact high levels of transcription can exasperate problems with downstream protein synthesis steps by overloading the cells capacity to produce and process these proteins. The use of selection systems applying selective pressure at an alternative stage of protein production can greatly improve the stringency of selection. Such selection systems can be termed orthogonal selection systems because their mode of action is independent of the current state of the art selection systems operating at the level of gene transcription. These alternative selection systems may also operate independently of each other and hence be orthogonal to each other. For example selection systems targeting transcription, translation, posttranslational modification and secretion might all be independent of each other in mode of action. Orthogonal selection systems can be used together to increase the stringency of selection and to hit multiple targets in selection.

The current invention is an example of an orthogonal selection system, operating at the level of protein folding and posttranslational modification. However other example of orthogonal selection systems can be envisioned working on the same broad principle of inhibiting the growth of cells within the population that perform poorly in a step of protein expression downstream of transcription.

In one aspect, the disclosure provides methods for generating a cell or cell line for producing a product, e.g., a recombinant polypeptide. In another aspect, the disclosure provides methods for producing a product, e.g., a recombinant polypeptide described herein using a cell that is identified, classified, selected, or generated using the methods described herein. Any of the foregoing methods include evaluating, identifying, classifying, or selecting a cell as described herein, e.g., by contacting the cell with an inhibitor of protein degradation, to identify or make a cell that has the capacity for high production of a product, e.g., a recombinant polypeptide. The methods described herein increase the production, e.g., expression and/or secretion of a recombinant polypeptide.

Without wishing to be bound by theory, it is believed that cells capable of higher productivity are less susceptible to inhibitors of protein degradation, and therefore, it is believed that contacting the cells with an inhibitor of protein degradation comprising an exogenous nucleic acid described herein results in the selection for a cell capable of higher productivity.

In one aspect, utilization of two or more selection steps, e.g., selection for stable integration of the exogenous nucleic acid and selection for susceptibility for inhibition of protein degradation, results in higher producing cells and cell lines than cells generated by a single selection step. Accordingly, the present disclosure features a method for generating a high producing cell comprising:

i) providing a cell that comprises an exogenous nucleic acid encoding a recombinant polypeptide product;

ii) administering a first selective pressure, e.g., selection for a first property, e.g., selecting for cells having stable integration of the exogenous nucleic acid; and

iii) administering a second selective pressure, e.g., performing a selection for a second property, e.g., selecting for cells having lower susceptibility to inhibition of protein degradation.

In embodiments, the first selective pressure is administered prior to, simultaneously with, or after the second selective pressure is administered. In one embodiment, the first selective pressure is administered prior to the second selective pressure. In one embodiment, the first selective pressure is completed prior to initiation of the second selective pressure. In one embodiment, the second selective pressure is administered prior to the first selective pressure. In one embodiment, the second selection pressure is completed prior to the initiation of the first selective pressure. In one embodiment, the first selection pressure and the second selection pressure is administered simultaneously. In one embodiment, the administration of the first and second selection pressure overlap fully or in part. In one embodiment, the second selection pressure is initiated 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 168 hours or more prior to the completion of the first selection pressure. In one embodiment, the second selection pressure is initiated 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 168 hours or more after the completion of the first selection pressure.

In an embodiment, the first selective pressure comprises selection for stable integration of the exogenous nucleic acid, e.g., a GS/MSX selection. In an embodiment, the second selective pressure comprises selection for susceptibility to inhibition of protein degradation comprises contacting the cells with an inhibitor of protein degradation. In an embodiment, an inhibitor of protein degradation is administered prior to, simultaneously with, or after a selection pressure comprising identification of cells comprising genomic integration of the exogenous nucleic acid. In an embodiment, the inhibitor of protein degradation is administered 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 168 hours or more after the completion of a prior selection step to identify cells comprising genomic integration of the exogenous nucleic acid.

The selective pressures are applied in a manner to select for cells with improved capacity to produce recombinant polypeptides, and/or with ability to produce recombinant polypeptides with improved quality. In one embodiment, each selective pressure, e.g., a first and/or second selective pressure described herein, is applied for at least 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours, or 168 hours. As described herein, the concentration of an inhibitor of protein degradation results in at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%

culture viability, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the number of cells remain, e.g., survive, after culturing in the presence of an inhibitor of protein degradation described herein. In some embodiments, the methods described herein, e.g., particularly after application of two or more selective pressures, results in less than 20%, e.g., 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, total cells remaining after the selection. The percentage of culture viability or cell survival after selection in the presence of an inhibitor of protein degradation described herein is determined as compared to the percentage of culture viability or cell survival in the absence of the presence of an inhibitor of protein degradation. The remaining viable cells after culture in the presence of inhibitor of protein degradation described herein can be further cultured to generate a cell line.

In embodiments, viable cells that remain after two selective pressures, e.g., selection for stable integrants comprising the exogenous nucleic acid and selection for low susceptibility to inhibitors to protein degradation, have increased capacity for protein production. For example, the cells having increased capacity for protein production produce 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more, or at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more of a product, e.g., a recombinant polypeptide, as compared to cells that were only administered one selective pressure or were not administered any selective pressure.

In embodiments, viable cells that remain after two selective pressures as described herein, produce an improved quality of the expressed recombinant polypeptide. For example, the improved quality of the product comprises one or more of: a more homogeneous product, an increased proportion of properly folded expressed recombinant polypeptides, or an increased proportion of functional recombinant polypeptides, a reduced proportion of aggregated recombinant polypeptides. For example, the cells producing improved quality of the product produce 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more, or at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, properly folded or functional recombinant polypeptides, as compared to cells that were only administered one selective pressure or were not administered any selective pressure.

In one embodiment, the methods used herein for generating a cell line with improved production capacity or improved quality of the produced product are useful with pre-existing or commercially available cell lines that express a recombinant polypeptide described herein, e.g.,

What is claimed is:

A method of evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for production of a product, e.g., a polypeptide, a recombinant polypeptide, comprising:

a) optionally, providing a cell;

b) contacting the cell (or progeny of the cell) with an inhibitor of protein degradation, e.g., a proteasome inhibitor, a ubiquitin pathway inhibitor, or an endoplasmic reticulum associated degradation (ERAD) inhibitor;

c) evaluating the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, ubiquitin pathway inhibitor, or an ERAD inhibitor, on one or more parameters related to cell function, in the cell (or progeny of the cell);

d) optionally, comparing a value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the one or more parameters with a reference value; and

e) optionally, expressing a product, e.g., a recombinant polypeptide from the cell (or progeny of the cell); and

thereby evaluating, classifying, identifying, making, or selecting, a cell or a progeny cell or population of progeny cells.

2. The method of claim 1 , further comprising, e.g., as a part of step (b), culturing the cell (or progeny of the cell) in contact with the inhibitor of protein degradation, e.g., to provide a population of cultured cells, e.g., a population of progeny cells.

3. The method of claim 1 or 2, comprising comparing a value for the effect of the inhibitor of protein degradation, e.g., a proteasome inhibitor, with a reference value, e.g., wherein when the value meets or exceeds the reference value, identifying, classifying, or selecting, a cell or a progeny cell or population of progeny cells, e.g., for establishing a culture, e.g., a cell bank or for production of a product, e.g., a polypeptide, e.g., a recombinant polypeptide.

4. The method of any of claims 1-3, further comprising establishing a cell line from the cell or a progeny cell.

5. The method of any of claims 1-4, wherein a parameter related to cell function comprises is selected from:

i) cell survival,

ii) culture viability,

iii) the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, iv) proteasome activity; or

v) quality of the expressed product, e.g., a polypeptide, e.g., a recombinant polypeptide, e.g., a more homogeneous product.

6. The method of any of claims 1-5, wherein the inhibitor of protein degradation is a proteasome inhibitor, optionally, wherein the proteasome inhibitor inhibits or reduces the activity of one or more of the 20S core subunit, the 19S regulatory subunit, the 1 I S regulatory particle, or a chaperone protein that assist proteasome assembly, e.g., Hsm3/S5b, Nas2/p27, Rpnl4/PAAF1, and Nas6/gankyrin, optionally, wherein the proteasome inhibitor is selected from MG132, epoxomicin, bortezomib, ixazomib, carfilzomib, disulfiram, CEP-18770, ONX 0912, salinosporamide, LLnV, CEP 1612, lactacystin, PS-341, and eponomicin.

7. The method of any of claims 1-5, wherein the inhibitor of protein degradation is an ERAD inhibitor, optionally, wherein the ERAD inhibitor inhibits or reduces the activity of one or more of calnexin/calreticulin, UDP-glucose-glycoprotein glucosyltransferase, ER degradation enhancing a-mannosidase-like protein (EDEM), ER mannosidase I, Sec61 , CDC48p (VCP/p97), Hrdl, DoalO, Ubc6, Ubcl , Cuel, or Ubc7, optionally, wherein the inhibitor of protein degradation is eeyarestatin I.

8. The method of any of claims 1-5, wherein the inhibitor of protein degradation is an ubiquitin pathway inhibitor, optionally, wherein the ubiquitin pathway inhibitor inhibits or reduces the activity of one or more of: El ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, or E3 ubiquitin ligase.

9. The method of any of claims 6-8, further comprising:

contacting the cell with a second inhibitor of protein degradation, e.g., a proteasome inhibitor, an ERAD inhibitor, or a ubiquitin pathway inhibitor, optionally, wherein the cell is contacted with a first and a second inhibitor of protein degradation, e.g., concurrently or sequentially.

10. The method of claim 9, wherein:

(i) the first inhibitor of protein degradation and second inhibitor of protein degradation are selected from MG132, epoxomicin, or eeyarestatin 1;

(ii) the first inhibitor is a proteasome inhibitor and the second inhibitor is a proteasome inhibitor;

(iii) the first inhibitor is a proteasome inhibitor and the second inhibitor is an ERAD inhibitor or an ubiquitin pathway inhibitor;

(iv) the first inhibitor is an ERAD inhibitor and the second inhibitor is an ERAD inhibitor;

(v) the first inhibitor is an ERAD inhibitor and the second inhibitor is an ubiquitin pathway inhibitor or a proteasome inhibitor;

(vi) the first inhibitor is an ubiquitin pathway inhibitor and the second inhibitor is an ubiquitin pathway inhibitor; or

(vii) the first inhibitor is an ubiquitin pathway inhibitor and the second inhibitor is an ERAD inhibitor or a proteasome inhibitor.

11. The method of any of claims 1-10, wherein the cell is contacted with a concentration of the inhibitor of protein degradation that is sufficient to reduce culture viability, by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, e.g., as compared to the culture viability before the culture is contacted with the inhibitor or as compared to a culture that is not contacted with the inhibitor, optionally, wherein the concentration of the inhibitor of protein degradation is a concentration at which one or more cells continue to proliferate.

12. The method of any of claims 1-11 , wherein the concentration of the inhibitor of protein degradation is less than 0.5 μΜ MG-132, e.g., about 0.0625 μΜ MG-132; or less than 0.05 μΜ epoxomicin, e.g., about 0.025 μΜ epoxomicin.

13. The method of any of claims 1 -12, wherein the cell is contacted by, e.g., cultured in, the inhibitor of protein degradation for 24, 48, 72, 96, or more hours.

14. The method of any of claims 1-13, wherein the cell is contacted by the inhibitor of protein degradation 24, 48, 72, 96, or 168 hours prior, after, or simultaneously or concurrently a selection step, e.g., MSX selection.

15. The method of any of claims 1-14, comprising:

providing a cell that expresses the product, e.g., the polypeptide, e.g., the recombinant polypeptide, e.g., a cell that comprises an exogenous nucleic acid that encodes the polypeptide, e.g., the recombinant polypeptide or controls the expression of the polypeptide, e.g., the recombinant polypeptide.

16. The method of claim 15, wherein the product comprises a recombinant polypeptide, e.g., an antibody molecule (e.g., a monoclonal antibody), a bispecific molecule, a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, or an enzyme, e.g., selected from Table 1 or 2.

17. The method of any of claims 1-16, wherein the cell is a eukaryotic cell, e.g., a mammalian cell.

18. The method of any of claims 1-17, wherein the cell is from mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, human, dog, camel, horse, ferret, or cat; or is a eukaryotic cell other than a mammalian cell, e.g., the cell is from an insect, plant, duck, parrot, fish, or yeast.

19. The method of any of claims 1-18, wherein the cell is a CHO cell, e.g., CHOK1, CHOKISV, Potelligent CHOKI SV, CHO GS knockout, CHOKISV GS-KO, CHOS, CHO DG44, CHO DXB11 , CHOZN, or a CHO-derived cell.

20. The method of any of claims 1-18, wherein the cell is a selected from Hela,

HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK, VERO, SP2/0, NSO, YB2/0, EB66, C127, L cell, COS, e.g., COS1 and COS7, QCl -3, CHOK1, CHOKISV, Potelligent CHOKI SV, CHO GS knockout, CHOKI SV GS-KO, CHOS, CHO DG44, CHO DXBl l . and CHOZN.

21. The method of any of claims 1-20, comprising expressing the product, e.g., the polypeptide, e.g., the recombinant polypeptide, from the cell or a progeny cell, optionally, wherein the expressed product, e.g., the polypeptide, e.g., the recombinant polypeptide, is evaluated for a parameter related to a physical or functional property, e.g., primary sequence, glycosylation, primary, secondary, tertiary, or quaternary structure, activity, degree of glycosylation, degree of aggregation, proportion or level of the expressed product having a preselected property, e.g., having a preselected monomeric, dimeric, or trimeric structure, or the level or proportion of the expressed product having a preselected structure, e.g., non-denatured or non-aggregated structure.

22. The method of any of claims 1-21 , further comprising introducing an exogenous nucleic acid into the cell, optionally, wherein the exogenous nucleic acid is introduced prior to or after one or more of steps a), b), c) and d).

23. The method of claim 22, wherein the exogenous nucleic acid encodes a polypeptide, e.g., a recombinant polypeptide, e.g. a therapeutic polypeptide or an antibody molecule selected from Table 1 or 2.

24. The method of claim 1 -23, further comprising introducing one or more additional exogenous nucleic acids, e.g., a second exogenous nucleic acid, into the cell, optionally, wherein introducing an exogenous nucleic acid comprises transfection, electroporation, or transduction, optionally, wherein the second exogenous nucleic acid is introduced prior to or after one or more of steps a), b), c) and d).

25. The method of claims 22-24, wherein the second exogenous nucleic acid is introduced prior to, after, or simultaneously with the introduction of the first exogenous nucleic acid, optionally, wherein the second exogenous nucleic acid encodes a selection marker, e.g., glutamine synthase.

26. The method of claims 1-25, further comprising evaluating the cell for a second property, e.g., determining if the cell comprises an exogenous component, e.g., one or more exogenous nucleic acids, e.g., one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell, optionally, wherein determining if the cell comprises one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell, comprises MSX selection, MTX selection (e.g., DHFR system), antibiotic selection, yeast growth selection, selection based on color change or surface expression of a marker, selection based on the Selexis system, or selection based on the Catalant system, optionally, wherein antibiotic selection comprises selection for resistance to an antibiotic selected from hygromycin, neomycin (G418), zeocin, puromycin, or blasticidin.

27. The method of any of claims 1 -26, wherein the cell comprises an exogenous nucleic acid that encodes the product, e.g., the recombinant polypeptide, or an exogenous nucleic acid that controls the expression of the product, e.g. endogenous polypeptide.

28. The method of any of claims 22-27, wherein the exogenous nucleic acid further comprises a nucleic acid sequence encoding a selection marker, optionally, wherein the selection marker comprises glutamine synthetase, DHFR, or a gene that confers antibiotic resistance.

29. The method of claim 28, wherein the nucleic acid sequence encoding the selection marker is operably linked to a first promoter and the nucleic acid sequence encoding the

polypeptide, e.g., the recombinant polypeptide is operably linked to a second promoter, optionally, wherein the first promoter and the second promoter are different.

30. The method of claim 29, wherein:

(i) the promoter operably linked to the selectable marker is a weak promoter, e.g., an SV40E promoter; and/or

(ii) the promoter operably linked to the nucleic acid sequence encoding the polypeptide, e.g., the recombinant polypeptide is a strong promoter, e.g., a CMV promoter, e.g., hCMV-MIE promoter.

31. The method of any of claims 1 -30, wherein the product, e.g., polypeptide, e.g., a recombinant polypeptide, has the same amino acid sequence or differs from a naturally occurring isoform of the human polypeptide at no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues, e.g., wherein the product, e.g., polypeptide, e.g., a recombinant polypeptide, is a polypeptide from Table 1 or Table 2.

32. The method of any of claims 1-31 , comprising:

c) evaluating the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on a parameter related to cell function, e.g., survival, viability, or the ability to proliferate, or to produce a product, e.g., a polypeptide, e.g., a polypeptide expressed from an exogenous nucleic acid; and

d) determining by, e.g., MSX selection, if the cell comprises an exogenous nucleic acid integrated into the chromosomal genome of the cell.

33. The method of any of claims 1-32, wherein the value for the effect of the inhibitor of protein degradation, e.g., proteasome inhibitor, on the parameter related to cell function exceeds the reference value by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or by 1 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold or more, wherein the reference value is the value of the effect of the inhibitor of protein degradation on a parameter related to cell function of a reference cell or the value of the parameter related to cell function of the cell that has not been contacted with the inhibitor of protein degradation.

34. The method of claim 33, wherein the parameter comprises one or more of:

(i) the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, e.g., a recombinant polypeptide expressed from an exogenous nucleic acid;

(ii) the ability to produce a product, e.g., a polypeptide, e.g., a recombinant polypeptide, and wherein the increase is determined by measuring or quantifying the product, e.g., the polypeptide, e.g., the recombinant polypeptide, in the cell or secreted by the cell;

(iii) cell survival, and wherein the increase or decrease is determined by measuring or quantifying the number of apoptotic cells; or

(iv) culture viability, and wherein the increase or decrease is determined by measuring or quantifying the number live cells.

35. The method of any of claims 1-34, further comprising an additional selection step, e.g., selection by FACS, magnetic separation (e.g., using magnetic beads), colony picking, micro fiuidic cell sorting, or microfiuidic cell destruction.

36. The method of any of claims 1-35, further comprising introducing to the cell an agent that assists in protein folding, e.g., chaperone molecules or small chemical molecules, optionally, wherein the agent that assists in protein folding comprises a nucleic acid encoding a chaperone protein or component of the protein folding pathway, e.g., XBPl , SRP14, BiP/GRP78, PDI, calnexin or cyclophilin B; or a small molecule chosen from DMSO, glycerol, or PBA.

37. A method of making a cell line, or a population of cells, comprising:

a) identifying or selecting a cell by the method of any of claims 1 -36; and

b) culturing the cell, or population of cells, to provide a cell line or population of cells.

38. The method of claim 37, wherein the identifying or selecting the cell comprises contacting the cell, or a population of cells, with an inhibitor of protein degradation, e.g., an inhibitor of protein degradation according to claims 6-12.

39. The method of either of claims 37 or 38, further comprising introducing to the cell an exogenous nucleic acid that encodes a product, e.g., a recombinant polypeptide, or an exogenous nucleic acid that controls, e.g., increases, the expression of a product, e.g., an endogenous polypeptide.

40. The method of any of claims 37-39, comprising expressing the product, e.g., the polypeptide, e.g., the recombinant polypeptide, from the cell or a progeny cell, optionally, wherein the expressed product, e.g., the polypeptide, e.g., the recombinant polypeptide, is evaluated for a parameter related to a physical or functional property, e.g., primary sequence, glycosylation, primary, secondary, tertiary, or quaternary structure, activity, degree of glycosylation, degree of aggregation, proportion or level of the expressed product having a preselected property, e.g., having a preselected monomeric, dimeric, or trimeric structure, or the level or proportion of the expressed product having a preselected structure, e.g., non-denatured or non-aggregated structure.

41. The method of any of claims 37-40, wherein the identifying or selecting a cell further comprises performing a selection step, e.g., a step for determining whether the cell comprises one or more exogenous nucleic acids, optionally, wherein the selection step comprises:

(i) determining whether the cell comprises one or more exogenous nucleic acids integrated into a nucleic acid of the cell, e.g., integrated into the chromosomal genome of the cell; or

(ii) MSX selection, MTX selection, antibiotic selection, yeast growth selection, selection based on color change or surface expression of a marker, selection based on the Selexis system, or selection based on the Catalant system.

42. The method of claim any of claims 37-41 , further comprising establishing a cell line from said cell.

43. The method of any of claims 37-42, wherein the product comprises a polypeptide, e.g., a recombinant polypeptide, e.g., comprises an antibody molecule (e.g., a monoclonal antibody), a bispecific antibody), a blood clotting factor, an anticoagulant, a hormone, an

interferon, an interleukin, or an enzyme, e.g., a polypeptide that has the same amino acid sequence or differs from a polypeptide from Table 1 or Table 2 by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

44. The method of any of claims 37-43, wherein the cell is a eukaryotic cell, e.g., a cell according to any of claims 17-20.

45. A method of making a product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprising:

a) providing a cell or a cell population made by the methods of any of claims 37-44; b) culturing the cell or a cell population in a culture medium; and

c) optionally, retrieving the product, e.g., polypeptide from the cells, cell population, or the culture medium.

46. The method of claim 45, comprising separating the polypeptide, e.g., the

recombinant polypeptide, from the cell or medium in which the cell was cultured.

47. The method of claim 45-46, wherein the product comprises an antibody molecule (e.g., a monoclonal antibody), a bispecific molecule, a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, or an enzyme, e.g., a polypeptide that has the same amino acid sequence or differs from a polypeptide from Table 1 or Table 2 by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

48. The method of any of claims 1-47, further comprising any of steps (f), (g), or both (f) and (g):

f) culturing the cell, or progeny of the cell, in the absence of the exogenous inhibitor of protein degradation (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages or for at least 12, 24, 48, 72 hours);

g) evaluating the level of recombinant protein produced by the cell, or progeny of the cell, e.g., to obtain a value of recombinant protein produced by the cell or progeny of the cell; and

h) optionally, comparing the value obtained in g) with a reference value, wherein the reference value is the level of recombinant protein produced by a cell of the same type as the cell provided in a) cultured in the absence of the exogenous inhibitor of protein degradation.

49. A method of making a cell, or progeny of cells, that produces a recombinant protein (e.g., a recombinant therapeutic protein, e.g., a recombinant therapeutic antibody), comprising: a) optionally, providing a cell;

b) culturing the cell or the progeny of the cell in the presence of an exogenous inhibitor of protein degradation, e.g., a proteasome inhibitor, a ubiquitin pathway inhibitor, or an endoplasmic reticulum associated degradation (ERAD) inhibitor (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more passages or for at least 12, 24, 48, 72 hours, or 1, 2, 4, 8, 16, 32 or more weeks);

c) culturing the cell or progeny of the cell in the absence of the exogenous inhibitor of protein degradation (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages or for at least 12, 24, 48, 72 hours);

d) optionally, evaluating the effect of the inhibitor of protein degradation on the level of recombinant protein produced by the cell or the progeny of the cell, e.g., to obtain a value of recombinant protein produced by the cell or the progeny of the cell; and

e) optionally, comparing the value obtained in d) with a reference value, wherein the reference value is the level of recombinant protein produced by a cell of the same type as the cell provided in a) cultured in the absence of the exogenous inhibitor of protein degradation;

thereby making the cell or progeny of cells.

50. The method of claim 49, further comprising f) selecting the cell for use in a method of manufacturing a recombinant protein (e.g., a recombinant therapeutic protein).

51. The method of claim 49 or 50, further comprising g) introducing an exogenous nucleic acid into the cell (e.g., after step c), after step d), after step e) or after step f)), optionally, wherein introducing the exogenous nucleic acid comprises transfection, electroporation, or transduction.

52. A cell, progeny cell, or population of progeny cells, evaluated, classified, selected, or made by the methods of any of claims 1 -51.

53. The cell of claim 52, wherein the cell is a eukaryotic cell, e.g., a cell according to any of claims 17-20.

54. The cell of either of claims 52 or 53, wherein the cell comprises an exogenous nucleic acid that controls e.g., increases, the expression of a product, e.g., a polypeptide, e.g., an endogenous polypeptide.

55. The cell of claim 54, wherein the product comprises a recombinant polypeptide, e.g., an antibody molecule (e.g., a monoclonal antibody or a bispecific molecule), a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, an enzyme, or a bispecific molecule, e.g., a polypeptide that has the same amino acid sequence or differs from a

polypeptide from Table 1 or Table 2 by no more than 1 , 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

56. A product, e.g., a polypeptide, e.g., a recombinant polypeptide, produced by or producible by the method of any of claims 1-51 , or the cell of any of claims 52-55.

57. The product, e.g., polypeptide, e.g., recombinant polypeptide, of claim 56, wherein the product, e.g., a polypeptide, e.g., a recombinant polypeptide, comprises an antibody molecule, a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, an enzyme, or a bispecific molecule, e.g., a polypeptide that has the same amino acid sequence or differs from a polypeptide from Table 1 or Table 2 by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues.

58. A preparation comprising a product, e.g., a polypeptide, e.g., a recombinant polypeptide, produced by, or producible by, the method of any of claims 1-51 , or the cell of any of claims 52-55, optionally, wherein at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the polypeptides in the preparation are properly folded or functionally active.

59. A mixture comprising a cell of any of claims 52-55, and the product, e.g., the polypeptide, e.g., recombinant polypeptide, wherein:

the product, e.g. the polypeptide, e.g., the recombinant polypeptide, is present at a higher concentration, e.g., at least, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher concentration, by weight or number, than would be seen in a cell that has not been contacted with inhibitor of protein degradation; or

wherein at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the product, e.g. the polypeptides, e.g., the recombinant polypeptides, in the mixture are properly folded or functionally active.

60. A preparation of medium conditioned by culture of a cell, progeny cell, or population of progeny cells, described herein, e.g., a cell, progeny cell, or population of progeny cells, of any of claims 52-55, wherein

the product, e.g., the polypeptide, e.g., the recombinant polypeptide, is present at a higher concentration, e.g., at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30% higher concentration, by weight or number, than would be seen in a cell that has not been contacted with an inhibitor of protein degradation; or

wherein at least 70, 80, 90, 95, 98 or 99 %, by weight or number, of the product e.g., the polypeptides, e.g., the recombinant polypeptides, in the mixture are properly folded or functionally active.

61. The preparation or mixture of any of claims 58-60, wherein the product comprises an antibody molecule, a blood clotting factor, an anticoagulant, a hormone, an interferon, an interleukin, an enzyme, or a bispecific molecule, e.g., has the same amino acid sequence or differs from a polypeptide from Table 1 or Table 2 by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues.