Method for reducing protease activities

Abstract

The present invention relates to mammalian cells modified to have reduced AEP protease activity. Such cells can be used for producing recombinant proteins.

Description

METHOD FOR REDUCING PROTEASE ACTIVITIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 678,169, filed August 1, 2024, and U.S. Provisional Patent Application Serial No. 63 / 846,898, filed on July 18, 2025.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in "xml" format and is identified by the file name 10985-W001-SEC_ SequenceListing, created July 24, 2025, which is 37,615 bytes in size. The subject matter contained in the electronic format of this sequence listing is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure relates to methods for reducing protease activities during recombination protein production process.BACKGROUND

[0004] In response to strong and growing demand for biological therapeutics, significant advances in bioprocessing have been made to facilitate cost-efficient, large-scale recombinant protein production. Illustratively, new and improved high cell density culture methods and intensified cell culture processes have enabled greater volumetric productivity at reduced cost. However, in addition to increasing protein titers, these upstream processes typically result in higher cell densities and process-related impurity7levels (e.g., host cell proteins (HCPs) and nucleic acids), which increase the burden on the costly downstream clarification and purification operations used to isolate recombinant proteins.

[0005] Cell culture clarification is a dow nstream unit operation in which cells, cellular debris, and other process-related impurities are removed from cell culture harvest fluid prior to further downstream purification steps, such as, e.g.. chromatographic separation processes. Mechanical separation (such as, e.g., centrifugation) followed by depth filtration is a common approach for clarifying cell cultures. To increase filtration throughput and improve separationperformance, many clarification processes incorporate a pretreatment step, such as acid precipitation or flocculant addition, that renders certain process-related impurities insoluble prior to mechanical separation of the remaining cell culture fluid (CCF).

[0006] However, as with all downstream unit operations, the process conditions used during cell harvesting must be carefully controlled to avoid product loss and product degradation. This is especially true when processing large scale cell biomasses due to the high concentration of lysosomal enzymes, such as cathepsins and pepsins, that may be secreted or released into the bioreactor during cell culture and harvest. Although lysosomal enzy mes are not active under the neutral pH conditions employed during cell culture, these enzymes may become active under acidic and mildly acidic pH conditions, such as those used during cell culture harvesting and protein purification.

[0007] Accordingly, there is a need in the art for new and improved cell culture harvesting methods that enable efficient clarification of high cell density cultures without inducing product cleavage by lysosomal enzymes that may be present within the cell culture.SUMMARY

[0008] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E).El . A method for producing a recombinant protein, wherein said recombinant protein comprises an asparaginyl endopeptidase (AEP) cleavage site, comprising:(a) culturing a mammalian cell culture under a condition wherein said recombinant protein is expressed, and wherein said mammalian cell is an AEP knockout cell;(b) harvesting said recombinant protein from the culture; and(c) purifying said recombinant protein, wherein said purification comprises a step wherein the recombinant protein is subject to an acidic environment with a pH of 5 or less.E2. The method of El, wherein said purification process comprises an acidic precipitation step.E3. The method of El or E2, wherein said harvesting in step (b) comprises harvesting the supernatant of the cell culture.E4. The method of E3, wherein the supernatant is harvested by cooling, flocculation, acidification, centrifugation, neutralization, acoustic wave separation, or filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration) of said cell culture.E5. The method of E3 or E4, wherein said purification in step (c) comprises adding an acidic solution to the supernatant, such that the pH of the resulting supernatant is 5 or less.E6. The method of E5, wherein the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing.E7. The method of any one of E1-E6, wherein said purification in step (c) comprises subjecting the recombinant protein to an acidic environment for at least about 60 minutes.E8. The method of any one of E1-E7, wherein said purification in step (c) comprises subjecting the recombinant protein to an acidic environment for about 60 minutes to about 48 hours.E9. The method of any one of E1-E8, wherein said purification in step (c) comprises an acidic precipitation step and a chromatography step, and the acidic precipitation step precedes the chromatography step.E10. The method of E9. wherein said chromatography is Protein A chromatography or Protein G chromatography.El l. The method of any one of El -El 0. wherein the recombinant protein is subject to an acidic environment with a pH of 4.6 or less.El 2. The method of any one of El -EE 11, wherein the recombinant protein is an immunoglobulin G (IgG).El 3. The method of any one of El -El 2, wherein the recombinant protein is an IgGl, IgG2, IgG3, or IgG4.E14. The method of any one of E1-E13, wherein the recombinant protein is an IgGEEl 5. The method of any one of El -El 3, wherein the recombinant protein is an IgG2.El 6. The method of any one of E1-E13, wherein the recombinant protein is abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab. eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, ontamalimab, ocrelizumab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, tafolecimab, tanezumab. tezepelumab, tremelimumab, utomilumab, or volagidemab.El 7. The method of El 6, wherein the recombinant protein is denosumab, erenumab, evolocumab, panitumumab, romosozumab, or tezepelumab.El 8. The method of El 6, wherein the recombinant protein is denosumab.El 9. The method of El 6, wherein the recombinant protein is erenumab.E20. The method of El 6, wherein the recombinant protein is evolocumab.E21. The method of El 6, wherein the recombinant protein is panitumumab.E22. The method of El 6, wherein the recombinant protein is romosozumab.E23. The method of El 6, wherein the recombinant protein is tezepelumab.E24. The method of El 6, wherein the recombinant protein is ocrelizumab.E25. The method of any one of E1-E24, wherein said recombinant protein comprises a P3-P2-P1-P1’-P2’-P3’ sequence motif, wherein Pl is Asn or Asp, Pl’ is any amino acid residue, and P2’ is a hydrophobic residue, and wherein the peptidyl bond between Pl and Pl ’ is cleaved by AEP.E26. The method of any one of E1-E25. wherein said recombinant protein comprises a P3-P2-P1-P1 ’-P2’-P3’ sequence motif, wherein Pl is Asn, Pl ’ is any amino acid residue, and P2’ is a hydrophobic residue selected from the group consisting of: glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).E27. The method of any one of E1-E26, wherein said recombinant protein comprises a - NLA- sequence motif, and wherein the peptidyl bond between N and L is cleaved by AEP.E28. The method of any one of E1-E27, wherein the mammalian cell is a CHO cell or HEK cell.E29. The method of any one of E1-E28, wherein the mammalian cell is a CHO cell.E30. The method of any one of E1-E29, wherein both alleles encoding AEP of the mammalian cell have been knocked out.E31. The method of any one of E1-E30, wherein AEP has been knocked out using CRISPR technology.E32. The method of any one of E1-E30, wherein AEP has been knocked out using zinc- finger technology.E33. The method of any one of E1-E32. wherein the AEP activity of the mammalian cell is reduced as compared to a control mammalian cell where AEP has not been knocked out.E34. The method of E33, wherein said AEP activity is measured by mass spectrometry or an immunoassay.E35. The method of E33, wherein said AEP activity' is measured by a cleavage assay.E36. The method of any one of E1-E35. wherein the AEP proenzyme quantity of the mammalian cell is reduced as compared to a control mammalian cell where AEP has not been knocked out.E37. The method of E36, wherein the AEP proenzyme quantity is measured by mass spectrometry or an immunoassay.E38. A mammalian cell wherein one or both alleles encoding asparaginyl endopeptidase (AEP) have been knocked out.E39. The mammalian cell of E38, wherein both alleles encoding AEP of the mammalian cell have been knocked out.E40. The mammalian cell of E38 or E39, wherein AEP has been knocked out using CRISPR technology.E41. The mammalian cell of E38 or E39, wherein AEP has been knocked out using using zinc-finger technology.E42. The mammalian cell of any one of E38-E41, for use to recombinantly produce a protein, wherein said recombinant protein comprises an asparaginyl endopeptidase (AEP) cleavage site.E43. The mammalian cell of any one of E38-E42, for use to recombinantly produce an immunoglobulin G (IgG).E44. The mammalian cell of E42 or E43, wherein the recombinant protein is an IgGl, IgG2, IgG3, or IgG4.E45. The mammalian cell of E42 or E43, wherein the recombinant protein is an IgGl.E46. The mammalian cell of E42 or E43, wherein the recombinant protein is an IgG2.E47. The mammalian cell of E42 or E43, wherein the recombinant protein is abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab. eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, ontamalimab, ocrelizumab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, tafolecimab, tanezumab, tezepelumab, tremelimumab, utomilumab, or volagidemab.E48. The mammalian cell of 47, wherein the recombinant protein is denosumab, erenumab, evolocumab, panitumumab, romosozumab, or tezepelumab.E49. The mammalian cell of 47. wherein the recombinant protein is denosumab.E50. The mammalian cell of 47, wherein the recombinant protein is erenumab.E51. The mammalian cell of 47. wherein the recombinant protein is evolocumab.E52. The mammalian cell of 47, wherein the recombinant protein is panitumumab.E53. The mammalian cell of 47. wherein the recombinant protein is romosozumab.E54. The mammalian cell of 47, wherein the recombinant protein is tezepelumab.E55. The mammalian cell of 47, wherein the recombinant protein is ocrelizumab.E56. The mammalian cell of any one of E48-E55, wherein said recombinant protein comprises a P3-P2-P1-P1’-P2’-P3’ sequence motif, wherein Pl is Asn or Asp. PE is any amino acid residue, and P2’ is a hydrophobic residue, and wherein the peptidyl bond between Pl and PE is cleaved by AEP.E57. The mammalian cell of any one of E48-E56. wherein said recombinant protein comprises a P3-P2-P1-PE-P2’-P3’ sequence motif, wherein Pl is Asn, PE is any amino acid residue, and P2‘ is a hydrophobic residue selected from the group consisting of: glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).E58. The mammalian cell of any one of E48-E57, wherein said recombinant protein comprises a -NLA- sequence motif, and wherein the peptidyl bond between N and L is cleaved by AEP.E59. The mammalian cell of any one of E48-E58, wherein the mammalian cell is a CHO cell or HEK cell.E60. The mammalian cell of any one of E48-E59, wherein the mammalian cell is a CHO cell.E61. The mammalian cell of any one of E48-E60, wherein the AEP activity of the mammalian cell is reduced as compared to a control mammalian cell where AEP has not been knocked out.E62. The mammalian cell of E61. wherein said AEP activity is measured by mass spectrometry or an immunoassay.E63. The mammalian cell of E61, wherein said AEP activity is measured by a cleavage assay.E64. The mammalian cell of E61, wherein the AEP proenzy me quantity of the mammalian cell is reduced as compared to a control mammalian cell where AEP has not been knocked out.E65. The mammalian cell of E61, wherein the AEP proenzyme quantity7is measured by mass spectrometry or an immunoassay.E66. A method for producing a mammalian cell, wherein one or both alleles encoding asparaginyl endopeptidase (AEP) have been knocked out, comprising:(a) introducing to said cell two guide RNAs (gRNAs). first one comprising single guide RNA1 (sgRNAl), and second one comprising sgRNA2, wherein: (i) sgRNAl is complementary7and binds to the antisense strand of AEP-encoding DNA and sgRNA2 is complementary7and binds to the sense strand of AEP-encoding DNA; (ii) the binding site for sgRNAl and sgRNA2 are 14 to 40 nucleotides apart; (3) an NGG sequence immediately follows the 3’-end of sgRNAl; and (4) an NGG sequence immediately follows the 3’-end of sgRNA2; and(b) introducing to said cell a nucleic acid encoding a deactivated Cas9 (dCas9) and a Clo51.E67. The method of E66, further comprising assessing AEP activity of the mammalian cell.E68. The method of E67, wherein the AEP activity of the mammalian cell is reduced, as compared to a control.E69. The method of E68, wherein said control is the AEP activity' of a wild type of mammalian cell, preferrable from the same cell line.E70. The method of any one of E67-E69, wherein said mammalian cell expresses an immunoglobulin that comprises an AEP cleavage site, and wherein said AEP activity7is assessed by measuring protease cleavage of said immunoglobulin.E71. The method of any one of E66-E70, wherein the binding site for sgRNAl and sgRNA2 are 16 to 30 nucleotides apart.E72. The method of any one of E66-E71, wherein said sgRNAl and sgRNA2 bind to exon 1 of the AEP-encoding DNA.E73. The method of any one of E66-E72, wherein sgRNAl comprises any one of SEQ IDNos: 34-38.E74. The method of any one of E66-E73, wherein sgRNA2 comprises any one of SEQ ID Nos: 34-38.E75. The method of any one of E66-E74, wherein sgRNAl and sgRNA comprises the sequence pairs as shown in Table 2.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows that reduced capillary electrophoresis with SDS (rCE-SDS) identified a small percentage of low molecular weight (LMW) species in IgGl antibody (mAbl).

[0010] FIGs. 2A-2B show two different purification processes. FIG. 2C shows the total host cell protein (HCP) levels at different purification steps. FIG. 2D shows the pH level at different purification steps.

[0011] FIG. 3 A shows that total HCP signal intensity in HCCF was decreased relative to supernatant after cell debris and HCPs were precipitated. FIG. 3B shows that AEP legumain intensity (quantified by top 3 peptides) remained consistent between the two samples indicating it was not removed by acid precipitation step. FIG. 3C shows that the abundance of the AEP autocleavage peptides were quantified to measure a small, but statistically significant decrease in HCCF as compared to supernatant. FIG. 3D shows the sequence of CHO AEP legumain. Peptides detected by LC-MS are shown in bold. Proenzy me cleavage sites are indicated by blue stars on the highlighted tryptic peptides.

[0012] FIG. 4 is a schematic illustration of AEP inhibitor study design.

[0013] FIG. 5A shows that the addition of Fmoc-Ala-Glu-Asn-Lys-NH2 (AENK) inhibitor decreased the mAbl -NJ'L- fragmentation and LMW (-NJ'L- clip fragment abundance) percentage in an inhibitor concentration dependent manner. Supernatant and HCCF samples were buffer exchanged to pH 4.0 and pH 5.2 buffers with (+) and without (-) an AEP inhibitor and LMW clips were monitored by reduced RP LC-MS. FIG. 5B illustrates that relative activity of AEP is pH dependent. AEPs were activated by incubating under different pH conditions overnight at 16C.

[0014] FIG. 6A shows the result of reduced reversed-phase LC-UV chromatograms of mAbl supernatant (Final day bioreactor supernatant) in pH 4.0 and 5.2 buffers with (+) and without (-) AEP inhibitor AENK. FIG. 6B shows that the LMW clip (light chain -N^L- clip fragment) abundance was monitored to track AEP induced cleavage.

[0015] FIG. 7A shows the result of reduced reversed-phase LC-UV chromatograms of mAbl HCCF material in pH 4.0 and 5.2 buffers with (+) and without (-) AEP inhibitor AENK. FIG. 7B shows the LMW clip LC (light chain -N^L- clip fragment) abundance was monitored to track AEP induced cleavage.

[0016] FIG. 8A shows the region of exon 1 of AEP (asparaginyl endopeptidase) targeted for Cas-CLOVER cutting. FIG. 8B shows percentage distribution of edits in population of clones. FIG. 8C shows the region of deletion for 4 clones as compared to WT (mAbl production cell line), deletions are indicated by dotted lines.

[0017] FIG. 9A shows grow th in terms of VCD in 15-day production run for mAbl production cell line (WT), mAbl knock out pool (KO Pool), and knock out clones. FIG. 9B shows comparable viabilities throughout the production run for WT, KO Pool, and knock out clones.

[0018] FIG.10 shows the result of LC-MS proteomics quantitation of AEP levels in WT supernatant, AEP KO pool, and 4 AEP KO clonal cell lines. WT GMP2 bioreactor HCCF had greatest level of AEP (data not shown here). Label free relative quantitation was performed by normalizing MS peak areas to a yeast reference protein glucose-6-phosphate dehydrogenase (G6PDH) spiked in at a concentration of 100 ng / mg. AEP genetic knockout pool cell lines showed on average 80% decrease in detectable AEP levels. Monoclonal AEP KO cell lines show ed complete knockout of AEP with no AEP detected above a 0.5 ng / mg limit of detection of the proteomics method used in this study.

[0019] FIG. 11 is a volcano plot comparing HCP levels in AEP KO Pooll to WT supernatant. Legumain (AEP) is the only HCP detected being significantly downregulated in the KO cell lines. Significance thresholds are defined as a fold change (FC) greater than 4x, and a P-value < 0.05 (indicated by horizontal and vertical dashed lines). No proteins were found to be significantly changed other than legumain (AEP) in the knockout cells, indicating the genetic knockout of AEP does not significantly alter the viability of the cells.Additionally, the production titers were found to be comparable to the WT cell lines (5.3 mg / mL).

[0020] FIGs. 12A-12B show the correlation plots of measured HCP levels in: (A) AEP KO Pooll HCP vs WT supernatant, and (B) AEP KO Clone 1 vs WT supernatant measured by LC-MS proteomics. The observed levels of all HCPs correlate well between the WT and KO cell lines, with the exception of legumain (AEP) shown as a red dot which falls well below the correlation trendline.

[0021] FIG. 13 shows the result of reduced intact mass MS intensity -based quantitation of LC CDR2 cleavage products in WT GMP2 Bioreactor HCCF (WT HCCF), WT PMCB Clone 203 (WT supernatant), AEP KO pool, and AEP KO clonal cell lines after holding at pH 4.6 for 1, 24, and 48 hours at room temperature. Relative quantitation of -N^L- cleavage fragments was calculated as the sum of the N and C-terminal fragments as a fraction of the total intact and fragmented LC. WT HCCF from large scale ATO GMP2 bioreactor run was found to have the highest levels of -NJ'L- cleavage fragmentation, followed by the WT supernatant from the smaller scale fed-batch run. Supernatant from the KO cell pools, and KO clonal cell lines had less than 0.5% of the -N^L- cleavage fragment detected at 48 hrs. The observed levels of -N^L- cleavage fragments after acid precipitation correlate well with the levels of AEP detected from the LC-MS proteomics results (FIG. 10).

[0022] FIGs. 14A-14D are graphs of deconvoluted mass spectra of C-terminal fragments of LC -N^L- cleavage products (from residue L to C-terminus) in WT and AEP KO cell lines after holding at pH 4.6 for 1. 24. and 48 hours at room temperature.

[0023] FIG. 15 shows the target DNA region where a gRNA pair are designed. CCN and NGG are two PAMs, “n” refers to nucleotides where gRNA should target (bind), and X is a spacer between two gRNA targeting sequences.DETAILED DESCRIPTION

[0024] As disclosed and exemplified herein, during the downstream purification of an IgGl monoclonal antibody (mAbl). the inventors identified a small percentage of low molecular weight (LMW) species. Subsequent studies showed that the LMW was caused by the presence of enzyme asparaginyl endopeptidase (AEP), also known as legumain, inbiomanufacturing process pools. In this particular example, the peptidyl bond between residues -NL-, from the antibody light chain, was clipped by AEP. It was further confirmed that AEP was activated by acidic environments.

[0025] Several solutions can be used to reduce the AEP activity, thereby increasing the yield of a recombinantly produced protein. First, a purification step that removes substantial amount of AEP can be used before subjecting the sample to an acidic environment. For example, a chromatography step, such as protein A chromatography, can be used before acid precipitation (see, e.g., FIG. 2A). Second, an AEP inhibitor can be added to reduce the AEP activity (see, Example 2). Finally, a genetically-engineered host cell, where endogenous AEP has been knocked out, can be used as a host cell to recombinantly produce a protein. A genetically-engineered host cell lacking AEP activities (or with reduced AEP activities) would be highly desirable as a long-term solution, and can potentially reduce the inconvenience and costs associated with modifying protein purification processes (e.g., including a chromatography step early on), or inclusion of an AEP inhibitor.Asparaginyl Endopeptidase (AEP)

[0026] “Asparaginyl endopeptidase;’ which is also referred to as “AEP,” “legumain.” and “5- secretase,” is a lysosomal cysteine endopeptidase from the Cl 3 peptidase family, which hydrolyzes substrates at the C-terminus of asparagine residues at mildly acidic pH (2.5 < pH < 4.5). AEP converts from an inactive proenzy me form to a mature, active form by autocleavage of terminal portions at mildly acidic pH. Zhao et al, Cell Research (2014) 24:344- 358. Chinese Hamster Ovary' (CHO) cell AEP is predicted to possess the following sequence, including signal peptide:MCRMIWKAAVLLSLALGAGALAVGVDDPEDAGKHWVVIVAGSNGW YNYRHQADACHAYQIIHRNGIPDEQIIVMMYDDIANSEDNPTPGIVINR PNGTDVYAGVLKDYTGEDVTPENFLAVLRGDAEAVKGKGSGKVLRS GPQDHVFVYFTDHGATGLLVFPNEDLHVKDLNKTIRYMYEHKMYQK MVFYIEACESGSMMNHLPNDINVYATTAANPHESSYACYYDEERNTY LGDWYSVNWMEDSDVEDLTKETLHKQYHLVKSHTNTSHVMQYGNK SISTMKVMQFQGMKHSTSSPISLPPVTRLDLTPSPEVPLTILKRKLMSTN DLKQSQNLVGQIQRLLDARHVIEKSVHKIVSLLAGFGETAERLLSERAVLMAHDCYQEAVTHFRTHCFNWHSPTYEYALRHLYVLANLCEKPYPIDRIKMAMDKVCLSHY (SEQ ID NO: 1)

[0027] AEP sequences of mouse (M. musculus), human (H. sapiens), and wild pig (S. scrofa) were aligned in Zhao et al, Cell Research (2014) 24:344-358, to create a universal numbering scheme for AEP enzymes. The sequence of CHO AEP is three residues longer due to three additional residues in the signal peptide at the N-terminus. Accordingly, CHO AEP residues N328, D308, and E314 in SEQ ID NO: 1 correspond to N325, D305, and E311 in the universal AEP numbering proposed by Zhao et al, Cell Research (2014) 24:344-358, for AEP from mouse, human, and wild pig.

[0028] In general, AEP hydrolyzes substrates at the C-terminus of asparagine residue (-Asn- Xaa- peptidyl bond). AEP may also hydrolyze substrates at the C-terminus of aspartic acid residue.

[0029] Amino acid residues surrounding the peptide cleavage site for substrates of AEP are commonly labelled following the numbering conventions adopted by Schechter and Berger for substrates of proteases (Schechter I. Berger A. On the size of the active site in proteases.I. Papain. Biochem. Biophys. Res. Commun. 1967;27: 157-162). From the cleavage site to the peptide N-terminus, amino acid residues are numbered in ascending order starting from Pl. On the other side of the cleavage site, amino acid residues are denoted with a single prime and numbered in ascending order towards the C-terminus starting with Pl'. For peptide ligation, the amino acid positions on the incoming peptide are labelled with a double prime and numbered in ascending order from the N-terminus starting with Pl'. Therefore, AEP substrate can be characterized as having the following motif:P3-P2-P1-P1 -P2’-P3’ wherein Pl is Asn (or Asp), PE is non-specific (any amino acid), and P2’ is hydrophobic residue. Cleavage occurs at the bond between Pl and Pl’. P2’ is preferably a large hydrophobic residue. However, large hydrophobic residue is not always required. As shown in the examples, the exemplary mAb comprises an AEP cleavage site that comprises the following motif: -PSNLA-, wherein the peptidyl bond between N and L is cleaved. Amino acids that have hydrophobic side chains are glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan(Trp). Large Hydrophobic Residues (LHR) generally refers to phenylalanine, isoleucine, leucine, methionine and valine.Host Cells

[0030] Cell lines (also referred to as ‘"cells” or “host cells”) used in the present disclosure are genetically engineered to express a recombinant protein of commercial or scientific interest. Cells may be suitable for adherent, monolayer, and / or suspension culture, transfection, and expression of recombinant proteins, such as, e.g., antibodies. The cells can be used, for example, with batch, fed batch, and perfusion or continuous culture methods. Such cells are typically cell lines obtained or derived from mammals and are able to grow and survive when placed in either monolayer culture or suspension culture in medium containing appropriate nutrients and / or other factors, such as those described herein. The cells are typically selected that can express and secrete proteins, or that can be molecularly engineered to express and secrete, large quantities of a particular protein, more particularly, a glycoprotein of interest, into the culture medium. The selection of an appropriate host cell for expressing a recombinant protein will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule. In some embodiments of the methods of the present disclosure, the host cell is a mammalian host cell.

[0031] Cell lines are typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time. The cells can contain introduced, e.g., via transformation, transfection, infection, or injection, expression vectors (constructs), such as plasmids and the like, that harbor coding sequences, or portions thereof, encoding the proteins for expression and production in the culturing process. Such expression vectors contain the necessary' elements for the transcription and translation of the inserted coding sequence. Methods which are well known to and practiced by those skilled in the art can be used to construct expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual , 4thedition Cold Spring Harbor Press, Plainview, N.Y. or any of the previous editions; F. M. Ausubel et al., 2013,Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, or any of the previous editions; Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990. all of which are incorporated herein for any purpose.

[0032] Suitable host cells include, but are not limited to, those that are commercially available, for example, from culture collections such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0033] In some embodiments, the host cells are CHO cells. CHO cells, including CHOK1 cells (ATCC CCL61). are widely used to produce complex recombinant proteins. In some embodiments, the dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cell lines (Kaufman R. J., 1990, Meth Enzymol 185:537-566). Also included are the glutamine synthase (GS)-knockout CHOK1SV cell lines, making use of glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells for use in a biomanufacturing process disclosed herein include, but are not limited to, the following (ECACC accession numbers in parenthesis): CHO (85050302); CHO (PROTEIN FREE) (00102307); CHO-K1 (85051005); CHO-K1 / SF (93061607); CHO / dhFr-(94060607); CHO / dhFr-AC-free (05011002); and RR-CHOKI (92052129).

[0034] In some embodiments, the engineered cell lines can be derived from human cell lines. Non-limiting examples of suitable human cell lines includes human embryonic kidney cells (HEK293, HEK293T); human connective tissue cells (HT-1080); human cervical carcinoma cells (HELA); human embryonic retinal cells (PER.C6); human kidney cells (HKB-11); human liver cells (Huh-7); human lung cells (W138); human liver cells (Hep G2); human U2- OS osteosarcoma cells, human A549 lung cells, human A-431 epidermal cells, or human K562 bone marrow cells.

[0035] To generate host cell lines (e.g., mammalian cell lines) engineered to express a recombinant protein of interest, one or more nucleic acids encoding the recombinant protein (or components thereof in the case of multi-chain proteins) is initially inserted into one or more expression vectors. Nucleic acid control sequences useful in expression vectors forexpression in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed protein can be secreted by the recombinant host cell, for more facile isolation of the recombinant protein from the cell, if desired. Vectors may also include one or more selectable marker genes to facilitate selection of host cells into which the vectors have been introduced. In some embodiments, vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964). Suitable mammalian expression vectors are known in the art and are also commercially available.

[0036] Typically, vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a native or heterologous signal peptide sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a poly adenylation sequence, a poly linker region for inserting the polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. Vectors may be constructed from a starting vector such as a commercially available vector, and additional elements may be individually obtained and ligated into the vector.AEP knockout cell lines

[0037] Another aspect of the present disclosure provides methods for preparing or engineering the cell lines having reduced or eliminated expression of AEP. Chromosomal sequences encoding AEP can be knocked-down or knocked-out using a variety of techniques. Tn general, the engineered cell lines are prepared using a targeting endonuclease-mediated genome modification process. Persons skilled the art understand that said engineered cell lines also can be prepared using site-specific recombination systems, random mutagenesis, or other methods known in the art.

[0038] In general, engineered cell lines are prepared by a method comprising introducing into a parental cell line of interest at least one targeting endonuclease or nucleic acid encodingsaid targeting endonuclease, wherein the targeting endonuclease is targeted to a chromosomal sequence encoding AEP. The targeting endonuclease recognizes and binds the specific chromosomal sequence and introduces a double-stranded break. In some embodiments, the double-stranded break is repaired by a non-homologous end-joining (NHEJ) repair process. Because NHEJ is error prone, a deletion, insertion, and / or substitution of at least one nucleotide may occur, thereby disrupting the reading frame of the chromosomal sequence such that no protein product is produced. In other embodiments, the targeting endonucleases can also be used to alter a chromosomal sequence via a homologous recombination reaction by co-introducing a polynucleotide having substantial sequence identity with a portion of the targeted chromosomal sequence. In such situations, the double-stranded break introduced by the targeting endonuclease is repaired by a homology-directed repair process such that the chromosomal sequence is exchanged with the polynucleotide in a manner that results in the chromosomal sequence being changed or altered (e.g., by integration of an exogenous sequence).

[0039] A variety of targeting endonucleases can be used to modify the chromosomal sequences encoding AEP. The targeting endonuclease can be a naturally-occurring protein or an engineered protein. Suitable targeting endonucleases include, without limit, zinc finger nucleases (ZFNs), CRISPR nucleases, transcription activator-like effector (TALE) nucleases (TALENs). meganucleases, chimeric nucleases, site-specific endonucleases, and artificial targeted DNA double strand break inducing agents.

[0040] In specific embodiments, the targeting endonuclease can be a pair of zinc finger nucleases (ZFNs). ZFNs bind to specific targeted sequences and introduce a double-stranded break into a targeted cleavage site. Typically, a ZFN comprises a DNA binding domain (i.e., zinc fingers) and a cleavage domain (i.e., nuclease), each of which is described below.

[0041] DNA binding domain. DNA binding domains or the zinc fingers can be engineered to recognize and bind to any nucleic acid sequence of choice. See, for example, Beerli et al. (2002) Nat. Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nat. Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Zhang et al. (2000) J. Biol. Chem. 275(43):33850-33860; Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. An engineered zinc fingerbinding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising doublet, triplet, and / or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6.534,261, the disclosures of which are incorporated by reference herein in their entireties. As an example, the algorithm of described in U.S. Pat. No. 6,453,242 can be used to design a zinc finger binding domain to target a preselected sequence. Alternative methods, such as rational design using a nondegenerate recognition code table may also be used to design a zinc finger binding domain to target a specific sequence (Sera et al. (2002) Biochemistry 41 :7074-7081). Publicly available web-based tools for identifying potential target sites in DNA sequences as well as designing zinc finger binding domains are known in the art. For example, tools for identifying potential target sites in DNA sequences can be found at zincfingertools.org. Tools for designing zinc finger binding domains can be found at zifit.partners.org / ZiFiT. (See also. Mandell et al. (2006) Nuc. Acid Res. 34:W 1 -W523; Sander et al. (2007) Nuc. Acid Res. 35:W599-W605.)

[0042] A zinc finger binding domain can be designed to recognize and bind a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length. In one embodiment, the zinc finger binding domain can be designed to recognize and bind a DNA sequence ranging from about 9 to about 18 nucleotides in length. In general, the zinc finger binding domains of the zinc finger nucleases used herein comprise at least three zinc finger recognition regions or zinc fingers, wherein each zinc finger binds 3 nucleotides. In one embodiment, the zinc finger binding domain comprises four zinc finger recognition regions. In another embodiment, the zinc finger binding domain comprises five zinc finger recognition regions. In still another embodiment, the zinc finger binding domain comprises six zinc finger recognition regions. A zinc finger binding domain can be designed to bind to any suitable target DNA sequence. See for example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated by reference herein in their entireties.

[0043] Exemplary methods of selecting a zinc finger recognition region include phage display and two-hybrid systems, which are described in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; WO 01 / 88197 and GB 2,338.237, each of which is incorporated by reference herein in its entirety. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02 / 077227, the entire disclosure of which is incorporated herein by reference.

[0044] Zinc finger binding domains and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in detail in, for example, U.S. Pat. No. 7,888,121, which is incorporated by reference herein in its entirety. Zinc finger recognition regions and / or multi -fingered zinc finger proteins can be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the disclosures of w hich are incorporated by reference herein in their entireties, for non-limiting examples of linker sequences of six or more amino acids in length. The zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers of the protein.

[0045] Cleavage domain. A zinc finger nuclease also includes a cleavage domain. The cleavage domain portion of the zinc finger nuclease can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., SI Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzy mes (or functional fragments thereol) can be used as a source of cleavage domains.

[0046] A cleavage domain also can be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity. Two zinc finger nucleases can be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease can comprise both monomers to create an active enzyme dimer. As used herein, an “active enzyme dimer” is an enzyme dimer capable of cleaving a nucleic acid molecule. The two cleavage monomers can be derived from thesame endonuclease (or functional fragments thereof), or each monomer can be derived from a different endonuclease (or functional fragments thereof).

[0047] When two cleavage monomers are used to form an active enzyme dimer, the recognition sites for the two zinc fingers are preferably disposed such that binding of the two zinc fingers to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing. As a result, the near edges of the recognition sites can be separated by about 5 to about 18 nucleotides. For instance, the near edges can be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. It will however be understood that any integral number of nucleotides or nucleotide pairs can intervene between two recognition sites (e g., from about 2 to about 50 nucleotide pairs or more). The near edges of the recognition sites of the zinc finger nucleases, such as for example those described in detail herein, can be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites.

[0048] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g.. Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example. U.S. Pat. Nos. 5,356,802; 5,436.150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91 :883-887; Kim et al. (1994b) J. Biol. Chem. 269:31978-31982. Thus, a zinc finger nuclease can comprise the cleavage domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Exemplary Type IIS restriction enzymes are described for example in International Publication WO 07 / 014,275, the disclosure of which is incorporated by reference herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure. See, for example. Roberts et al. (2003) Nucleic Acids Res. 31 :418-420.

[0049] An exemplar}' Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fokl. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). Accordingly, for the purposes of the present disclosure, the portion of the Fokl enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double-stranded cleavage using a Fokl cleavage domain, two zinc finger nucleases, each comprising a Fokl cleavage monomer, can be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fokl cleavage monomers can also be used.

[0050] In certain embodiments, the cleavage domain comprises one or more engineered cleavage monomers that minimize or prevent homodimerization. By way of non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fokl are all targets for influencing dimerization of the Fokl cleavage half-domains. Exemplary engineered cleavage monomers of Fokl that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of Fokl and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499.

[0051] Thus, in one embodiment of the engineered cleavage monomers, a mutation at amino acid position 490 replaces Glu (E) with Lys (K); a mutation at amino acid residue 538 replaces Iso (I) with Lys (K); a mutation at amino acid residue 486 replaces Gin (Q) with Glu (E); and a mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage monomers can be prepared by mutating positions 490 from E to K and 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer designated “E490K:L538K” and by mutating positions 486 from Q to E and 499 from I to K in another cleavage monomer to produce an engineered cleavage monomer designated "Q486E: I499K." The above described engineered cleavage monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. Engineered cleavage monomers can be prepared using a suitable method, for example, by site-directed mutagenesis of wild-type cleavage monomers (Fokl) as described in U.S. Pat. No. 7,888,121, which is incorporated herein in its entirety7.

[0052] Additional domains. In some embodiments, the zinc finger nuclease further comprises at least one nuclear localization sequence (NLS). A NLS is an amino acid sequence whichfacilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO:2), PKKKRRV (SEQ ID NO:3), KRPAATKKAGQAKKKK (SEQ ID NO:4), YGRKKRRQRRR (SEQ ID NO:5), RKKRRQRRR (SEQ ID NO:6), PAAKRVKLD (SEQ ID NO:7). RQRRNELKRSP (SEQ ID NO: 8), VSRKRPRP (SEQ ID NOV), PPKKARED (SEQ ID NO: 10). PQPKKKPL (SEQ ID NO: 1 1), S ALIKKKKKMAP (SEQ ID NO: 12), PKQKKRK (SEQ ID NO: 13), RKLKKKIKKL (SEQ ID NO : 14), REKKKFLKRR (SEQ ID NO : 15), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 16), RKCLQAGMNLEARKTKK (SEQ ID NO: 17), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 18), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 19). The NLS can be located at the N-terminus, the C-terminus, or in an internal location of the zinc finger nuclease.

[0053] In additional embodiments, the zinc finger nuclease can also comprise at least one cell-penetrating domain. Examples of suitable cell-penetrating domains include, without limit, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO 20), PLSSIFSRIGDPPKKKRKV (SEQ ID NO:21), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO:22), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:23), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 24), YARAAARQARA (SEQ ID NO:25), THRLPRRRRRR (SEQ ID NO:26), GGRRARRRRRR (SEQ ID NO:27), RRQRRTSKLMKR (SEQ ID NO 28), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:29), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:30). and RQIKIWFQNRRMKWKK (SEQ ID NO:31). The cell-penetrating domain can be located at the N-terminus, the C-terminus, or in an internal location of the zinc finger nuclease.

[0054] In still other embodiments, the zinc finger nuclease can further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the marker domain can be a fluorescent protein. Non limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP. turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green. CopGFP. AceGFP. ZsGreenl), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g.EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan). red fluorescent proteins (mKate. mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed- Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira- Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In another embodiment, the marker domain can be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, poly(His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD), E tag. E2 tag. ECS tag, eXact tag, Glu-Glu tag, glutathione-S- transferase (GST), HA tag, HSV tag, KT3 tag, maltose binding protein (MBP). MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, SI tag, SBP tag, Softag 1 tag, Softag 3 tag. Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag, thioredoxin (TRX), V5 tag, VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, the C- terminus. or in an internal location of the zinc finger nuclease.

[0055] The at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one marker domain can be linked directly to the zinc finger nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, the at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one marker domain, can be linked indirectly to the zinc finger nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-am inobenzyloxycarbonyl, and the like), disulfide linkers, and polymer linkers (e.g., PEG). The linker can include one or more spacing groups including, but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker can be neutral, or carry a positive or negative charge. Additionally, the linker can be cleavable such that the linker's covalent bond that connects the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, a catalyst, or an enzyme. In some embodiments, the linker can be a peptide linker. The peptide linker can be a flexible amino acid linker or a rigid amino acid linker. Additional examples of suitable linkers are well known in the art and programs to design linkers are readily available (Crasto et al.. Protein Eng., 2000, 13(5):309- 312).

[0056] In other embodiments, the targeting endonuclease can be a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) nuclease. CRISPR nucleases are RNA- guided nucleases derived from bacterial or archaeal CRISPR / CRISPR-associated (Cas) systems. A CRISPR RNP system comprises a CRISPR nuclease and a guide RNA.

[0057] In general, CRISPR method comprises the following steps: (1) providing a cell line of interest, such as a human or CHO cell line; (2) designing and synthesizing a guide RNA (gRNA) that is complementary to a target sequence in the gene to be knocked out; (3) introducing the gRNA and a CRISPR-associated endonuclease (Cas) into the cell line, either by transfection, viral infection, electroporation, microinjection, or other suitable methods; (4) allowing the gRNA and the Cas to form a complex and bind to the target sequence in the gene to be knocked out; (5) allowing the Cas to cleave the target sequence, creating a doublestrand break (DSB) in the gene; (6) allowing the cell to repair the DSB by non-homologous end joining (NHEJ) or homology-directed repair (HDR), resulting in a mutation that disrupts the expression or function of the gene; and (7) selecting and isolating the cell line with the desired mutation, either by using a selectable marker, such as an antibiotic resistance gene, or by using a screening method, such as PCR, sequencing, or functional assay.

[0058] Nuclease. The CRISPR nuclease can be derived from a type I (i.e., IA, IB, IC, ID, IE, or IF), type II (i.e., IIA, IIB, or IIC), type III (i.e., IIIA or IIIB), type V, or type VI CRISPR system, which are present in various bacteria and archaea. For example, the CRISPR nuclease can be from Streptococcus sp. (e.g., S. pyogenes, S. thermophilus, S. pasteurianus), Campylobacter sp. (e.g., Campylobacterjejuni), Francisella sp. (e g., Francisella novicida), Acaryochloris sp., Acetohalobium sp.. Acidaminococcus sp., Aci dithiobacillus sp.. Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp.. Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp., Ktedonobacter sp., Lachnospiraceae sp.. Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscilla sp.. Microcoleus sp.. Microcystis sp., Natranaerobius sp., Neisseria sp.. Nitrosococcus sp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oscillatoria sp.,Polaromonas sp., Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp.. Streptomyces sp., Streptosporangium sp., Synechococcus sp., Thermosipho sp., or Verrucomicrobia sp. In other embodiments, the CRISPR nuclease can be derived from anarchaeal CRISPR system, a CRISPR / CasX system, or a CRISPR / CasY system (Burstein et al., Nature. 2017, 542(7640):237-241).

[0059] In some embodiments, the CRISPR nuclease can be derived from a type II CRISPR nuclease. For example, the type II CRISPR nuclease can be a Cas9 protein. Suitable Cas9 nucleases include Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus (SaCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9(CjCas9), Neisseria meningitis Cas9 (NmCas9), or Neisseria cinerea Cas9 (NcCas9). In other embodiments, the CRISPR nuclease can be derived from a type V CRISPR nuclease, such as a Cpfl nuclease. Suitable Cpfl nucleases include Francisella novicida Cpfl(FnCpfl), Acidaminococcus sp. Cpfl (AsCpfl), or Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl). In yet another embodiment, the CRISPR nuclease can be derived from a type VI CRISPR nuclease, e.g., Leptotrichia wadei Casl3a (LwaCasl3a) or Leptotrichia shahii Casl3a (LshCasl3a).

[0060] The CRISPR nuclease can be a wild type CRISPR nuclease, a modified CRISPR nuclease, or a fragment of a wild type or modified CRISPR nuclease. The CRISPR nuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR nuclease can be modified, deleted, or inactivated. The CRISPR nuclease can be truncated to remove domains that are not essential for the function of the nuclease.

[0061] CRISPR nucleases comprise two nuclease domains. For example, a Cas9 nuclease comprises a HNH domain, which cleaves the guide RNA complementary' strand, and a RuvC domain, which cleaves the non-complementary strand; a Cpfl nuclease comprises a RuvC domain and a NUC domain; and a Cas 13a nuclease comprises two HNEPN domains. When both nuclease domains are functional, CRISPR nuclease introduces a double-stranded break. Either nuclease domain can be inactivated by one or more mutations and / or deletions, thereby creating a variant that introduces a single-strand break in one strand of the double-stranded sequence. For example, one or more mutations in the RuvC domain of Cas9 nuclease (e.g., D10A, D8A, E762A, and / or D986A) results in an HNH nickase that nicks the guide RNA complementary' strand; and one or more mutations in the HNH domain of Cas9 nuclease(e.g., H840A, H559A, N854A, N856A, and / or N863A) results in a RuvC nickase that nicks the guide RNA non-complementary strand. Comparable mutations can convert Cpfl and Casl3a nucleases to nickases. Two CRISPR nickases targeted to opposites strands of a chromosomal sequence (via a pair of offset guide RNAs) can be used in combination to create a double-stranded break in the chromosomal sequence. Dual CRISPR nickase RNPs can increase target specificity and reduce off target effects.

[0062] Additional domains. The CRISPR nuclease can further comprise at least one nuclear localization sequence (NLS). A NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome. Nuclear localization signals are known in the art (see, e.g.. Lange et al., J. Biol. Chem., 2007, 282:5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO:2), PKKKRRV (SEQ ID NO:3), KRPAATKKAGQAKKKK (SEQ ID NO:4), YGRKKRRQRRR (SEQ ID NO:5), RKKRRQRRR (SEQ ID NO:6). PAAKRVKLD (SEQ ID NO:7). RQRRNELKRSP (SEQ ID NO:8), VSRKRPRP (SEQ ID NO:9), PPKKARED (SEQ ID NO: 10), PQPKKKPL (SEQ ID NO: 11), SALIKKKKKMAP (SEQ ID NO: 12), PKQKKRK (SEQ ID NO: 13), RKLKKKIKKL (SEQ ID NO : 14), REKKKFLKRR (SEQ ID NO : 15), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 16), RKCLQAGMNLEARKTKK (SEQ ID NO: 17), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 18), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 19). The NLS can be located at the N-terminus, the C-terminus, or in an internal location of the CRISPR nuclease.

[0063] In additional embodiments, the CRISPR nuclease can also comprise at least one cellpenetrating domain. Examples of suitable cell -penetrating domains include, without limit, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO 20), PLSSIFSRIGDPPKKKRKV (SEQ ID NO:21), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO:22), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:23), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:24), YARAAARQARA (SEQ ID NO:25), THRLPRRRRRR (SEQ ID NO:26), GGRRARRRRRR (SEQ ID NO:27), RRQRRTSKLMKR (SEQ ID NO 28), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:29), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:30). andRQIKIWFQNRRMKWKK (SEQ ID N0:31). The cell-penetrating domain can be located at the N-terminus, the C-terminus, or in an internal location of the CRISPR protein.

[0064] In still other embodiments, the CRISPR nuclease can further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the marker domain can be a fluorescent protein. Non limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. EBFP, EBFP2. Azurite, mKalamal. GFPuv, Sapphire, T-sapphire). cyan fluorescent proteins (e.g. ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed- Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira- Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In another embodiment, the marker domain can be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, poly (His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD). E tag. E2 tag. ECS tag. eXact tag, Glu-Glu tag, glutathione-S- transferase (GST), HA tag, HSV tag, KT3 tag, maltose binding protein (MBP), MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, SI tag, SBP tag, Softag 1 tag, Softag 3 tag, Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag. thioredoxin (TRX), V5 tag. VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, the C- terminus, or in an internal location of the CRISPR nuclease.

[0065] The at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one marker domain can be linked directly to the CRISPR nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, the at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one marker domain, can be linked indirectly to the CRISPR nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives. N-ethoxybenzylimidazole. biphenyl-3,4',5-tncarboxylic acid, p-am inobenzyloxy carbonyl, and the like), disulfide linkers, and polymer linkers (e.g., PEG). Thelinker can include one or more spacing groups including, but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl. aralkynyl and the like. The linker can be neutral, or carry a positive or negative charge. Additionally, the linker can be cleavable such that the linker's covalent bond that connects the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, a catalyst, or an enzyme. In some embodiments, the linker can be a peptide linker. The peptide linker can be a flexible amino acid linker or a rigid amino acid linker. Additional examples of suitable linkers are well known in the art and programs to design linkers are readily in the art.

[0066] Guide RNA. A CRISPR nuclease is guided to its target site by a guide RNA. The guide RNA hybridizes with the target site and interacts with the CRISPR nuclease to direct the CRISPR nuclease to the target site in the chromosomal sequence. The target site has no sequence limitation except that the sequence is bordered by a protospacer adjacent motif (PAM). PAM is typically 2-6 base pairs long) that is required for Cas proteins to recognize and bind to the target DNA. Without a PAM, Cas enzymes (like Cas9) cannot cleave the DNA, even if the guide RNA matches the target sequence perfectly. CRISPR proteins from different bacterial species recognize different PAM sequences. For example, PAM sequences include 5'-NGG (SpCas9, FnCAs9), 5 -NGRRT (SaCas9), 5'-NNAGAAW (StCas9). 5'- NNNNGATT (NmCas9), 5-NNNNRYAC (CjCas9), and 5'-TTTV (Cpfl), wherein N is defined as any nucleotide, R is defined as either G or A, W is defined as either A or T, Y is defined an either C or T, and V is defined as A, C, or G. Cas9 PAMs are located 3' of the target site, and cpfl PAMs are located 5' of the target site.

[0067] A guide RNA comprises three regions: a first region at the 5' end that is complementary' to sequence at the target site, a second internal region that forms a stem loop structure, and a third 3' region that remains essentially single-stranded. The first region of each guide RNA is different such that each guide RNA guides a CRISPR nuclease to a specific target site. The second and third regions (also called the scaffold region) of each guide RNA can be the same in all guide RNAs.

[0068] The first region of the guide RNA is complementary to sequence protospacer sequence) at the target site such that the first region of the guide RNA can base pair with sequence at the target site. The complementarity' between the first region (i.e., crRNA) of theguide RNA and the target sequence can be at least 80%, at least 85%, at least 90%. at least 95%. or more. In general, there are no mismatches between the sequence of the first region of the guide RNA and the sequence at the target site (i.e., the complementarity is total). In various embodiments, the first region of the guide RNA can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the first region of the guide RNA and the target site in the chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20. 22. 23. 24. 25. or more than 25 nucleotides in length. In exemplary embodiments, the first region of the guide RNA is about 19, 20, or 21 nucleotides in length.

[0069] The guide RNA also comprises a second region that forms a secondary structure. In some embodiments, the secondary structure comprises a stem (or hairpin) and a loop. The length of the loop and the stem can vary. For example, the loop can range from about 3 to about 10 nucleotides in length, and the stem can range from about 6 to about 20 base pairs in length. The stem can comprise one or more bulges of 1 to about 10 nucleotides. Thus, the overall length of the second region can range from about 16 to about 60 nucleotides in length. In an exemplary embodiment, the loop is about 4 nucleotides in length and the stem comprises about 12 base pairs.

[0070] The guide RNA also comprises a third region at the 3' end that remains essentially single-stranded. Thus, the third region has no complementarity to any chromosomal sequence in the cell of interest and has no complementarity to the rest of the guide RNA. The length of the third region can vary. In general, the third region is more than about 4 nucleotides in length. For example, the length of the third region can range from about 5 to about 60 nucleotides in length.

[0071] The combined length of the second and third regions (or scaffold) of the guide RNA can range from about 30 to about 120 nucleotides in length. In one aspect, the combined length of the second and third regions of the guide RNA range from about 70 to about 100 nucleotides in length.

[0072] In some embodiments, the guide RNA comprises one molecule comprising all three regions. In other embodiments, the guide RNA can comprise two separate molecules. The first RNA molecule can comprise the first (5') region of the guide RNA and one half of the “stem” of the second region of the guide RNA. The second RNA molecule can comprise theother half of the ‘‘stem” of the second region of the guide RNA and the third region of the guide RNA. Thus, in this embodiment, the first and second RNA molecules each contain a sequence of nucleotides that are complementary to one another. For example, in one embodiment, the first and second RNA molecules each comprise a sequence (of about 6 to about 20 nucleotides) that base pairs to the other sequence to form a functional guide RNA.

[0073] Cas-CLOVER. Cas-CLOVER is a modified CRISPR-Cas9 system that has demonstrated a lack of off-target activity associated with CRISPR-Cas9. Cas-CLOVER makes use of a catalytically inactive Cas9 protein, or “dead Cas9” (abbreviated as dCas9), fused to the dimeric Clo051 (also referred as Clo51) endonuclease domain, to catalyze the formation of a double stranded break in a target nucleic acid resulting in homologous recombination of a donor nucleic acid at the target site. Wild type Clo051 endonuclease sequence can be found, e.g., NCBI Reference Sequence WP 008676092.1, derived from the genome of Clostridium spec.7_2_43FAA. dCas9 is a modified version of the Cas9 protein from the CRISPR-Cas9 system that can still bind to DNA but cannot cut it. For example, D10A mutation of Cas9 inactivates the RuvC domain, and H840A mutation of Cas9 inactivates the HNH domain. The crystal structure of dCas9 in complex with gRNA and target DNA has been published (PDB ID 6K57). Because it is inactivated, dCas9 acts as a programmable DNA-binding protein, and is often fused to effector proteins, enabling targeted regulation or labeling of genomic regions.

[0074] Unlike Cas9, which is a single-component nuclease, Clo051 in general requires dimerization to become catalytically active. When used in synthetic gene editing platforms like Cas-CLOVER, Clo051 is fused to dCas9 to form a programmable nuclease system with enhanced specificity. In this setup, two dCas9-Clo051 fusion proteins are guided to adjacent DNA sites by paired guide RNAs. Only when both fusion proteins are properly positioned does Clo051 dimerize and introduce a double-stranded DNA break (DSB). This design significantly reduces off-target effects and makes Clo051 a preferred choice for high-fidelity genome editing, particularly in therapeutic or biomanufacturing settings where precision is critical.

[0075] Two gRNAs can be designed to target genes of interest to create double-stranded breaks, like other dimeric gene editing technologies (i.e. ZFN and TALEN). The Cas- CLOVER system requires two Protospacer Adjacent Motif (PAM) sequences and a flexiblespacer (preferred range of 11-40 nucleotides) for the dual-complex to function. This enables high fidelity gene editing with no detectable off-target activity when screened via nextgeneration sequencing (NGS) reads, while also maintaining flexibility to target any gene of interest. Examples of PAM sequence for Cas-CLOVER (SpCas9-based) include: canonical PAM: 5'-NGG, wherein N is any base (e g., AGG, TGG, GGG, CGG); extended PAM: NAG, NGA, NAA, wherein N is any base. The extended PAM is less efficient and Cas9 may bind weakly, so sometimes it may be desirable to avoid using extended PAM for precise editing.

[0076] Further details of the Cas-CLOVER system are provided in U.S. Patent Publication US2018 / 0187185 and International Publication WO 2024 / 007010, which are incorporated herein by reference in their entirety.

[0077] Designing guide-RNA pairs for Cas-CLOVER. A variety of open source gRNA design tools are available online for you: platforms that accommodate gRNA designs for paired Cas9 Nickase such as ZiFit, CHOPCHOP or Benchling work well for Cas-CLOVER designs. The two guide-RNAs should be designed in the PAMs-out orientation, separated by a spacer region of 11 - 40 nucleotides. Preferred ranges for spacers include: 14-40 nucleotide long, 16-40 nucleotide long, or 11-36 nucleotide long. In some embodiments, the spacer is 16-30 nucleotide long; in some embodiments, the spacer is 16-20 nucleotide long. The gRNA pairs also need to be in the PAMs-out orientation. The recruited Clo051 nuclease domains dimerize and introduces a single double-stranded break in this spacer region between the two gRNA target sites.

[0078] Full length gRNA in general is a fusion of the crRNA (CRISPR RNA, which comprises the sequence complementary to the target DNA) and tracrRNA (trans-activating crRNA, which binds to the crRNA and helps recruit Cas9 / dCas9) fusing into a single RNA molecule). Full length gRNA is typically about 100 nucleotides long.

[0079] In some embodiments, the design of the gRNA pair (designated as sgRNAl and sgRNA2, respectively, referring to sequences that correspond to the DNA target, not tracrRNA portion) follow the following rules: (1) both sgRNAs should immediately precede PAM (based on 5’-3?direction); (2) they must bind to opposite strands of DNA (i.e.. sgRNAl binds to the 3 ’-5’ strand; and sgRNA2 binds to the 5’-3’ stand); and (3) the DNA sequences that sgRNAl binds and sgRNA2 binds are separated by a spacer of 14-39 nucleotide long. By way of illustration, the following example shows the design of the gRNA pair, with dashlines indicating nucleotides that correspond to the target DNA sequences, and 5’-NGG as PAM (underlined):5’ CCN - [[sgRNAl]]NGG 3’ (5’-3’ DNA strand)3’ GGN[[sgRNA2]] - NCC- 5’ (3 -5’ DNA strand)The design rationale of gRNA pairs is also summarized as FIG. 15. Because the sgRNA pair need to be in the “PAMs-out” orientation, sgRNAl will bind to 3’-5’ DNA strand, and sgRNA2 will bind to 5 ’-3’ DNA strand.

[0080] In further embodiments, the targeting endonuclease can be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by long recognition sequences, i.e., the recognition sequence generally ranges from about 12 base pairs to about 40 base pairs. As a consequence of this requirement, the recognition sequence generally occurs only once in any given genome. Among meganucleases, the family of homing endonucleases named LAGLIDADG has become a valuable tool for the study of genomes and genome engineering (see, e.g., Amould et al., 2011, Protein Eng Des Sei, 24(l-2):27-31). Other suitable meganucleases include I-Crel and I-Dmol. A meganuclease can be targeted to a specific chromosomal sequence by modifying its recognition sequence using techniques well known to those skilled in the art.

[0081] In additional embodiments, the targeting endonuclease can be a transcription activator-like effector (TALE) nuclease. TALEs are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA targets. TALEs or truncated versions thereof may be linked to the catalytic domain of endonucleases such as FokI to create targeting endonuclease called TALE nucleases or TALENs (Sanjana et al., 2012, Nat Protoc, 7(1): 171-192) and Amould et al., 2011. Protein Engineering. Design & Selection, 24(l-2):27-31).

[0082] In alternate embodiments, the targeting endonuclease can be chimeric nuclease. Nonlimiting examples of chimeric nucleases include ZF-meganucleases, TAL-meganucleases, Cas9-Fokl fusions. ZF-Cas9 fusions, TAL-Cas9 fusions, and the like. Persons skilled in the art are familiar with means for generating such chimeric nuclease fusions.

[0083] In still other embodiments, the targeting endonuclease can be a site-specific endonuclease. In particular, the site-specific endonuclease can be a “rare-cutter”endonuclease whose recognition sequence occurs rarely in a genome. Alternatively, the sitespecific endonuclease can be engineered to cleave a site of interest (Friedhoff et al., 2007, Methods Mol Biol 352: 1 110123). Generally, the recognition sequence of the site-specific endonuclease occurs only once in a genome. In alternate further embodiments, the targeting endonuclease can be an artificial targeted DNA double strand break inducing agent.

[0084] The method comprises introducing the targeting endonuclease into the parental cell line of interest. The targeting endonuclease can be introduced into the cells as a purified isolated protein or as a nucleic acid encoding the targeting endonuclease. The nucleic acid can be DNA or RNA. In embodiments in which the encoding nucleic acid is mRNA, the mRNA may be 5' capped and / or 3' polyadenylated. In embodiments in which the encoding nucleic acid is DNA, the DNA can be linear or circular. The nucleic acid can be part of a plasmid or viral vector, wherein the encoding DNA can be operably linked to a suitable promoter. Those skilled in the art are familiar with appropriate vectors, promoters, other control elements, and means of introducing the vector into the cell of interest. In embodiments in which targeting endonuclease is a CRISPR nuclease, the CRISPR nuclease system can be introduced into the cell as a gRNA-protein complex.

[0085] The targeting endonuclease molecule(s) can be introduced into the cell by a variety of means. Suitable delivery means include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes. or artificial virions. In a specific embodiment, the targeting endonuclease molecule(s) are introduced into the cell by nucleofection.

[0086] Optional Donor Polynucleotide. The method for targeted genome modification or engineering can further comprise introducing into the cell at least one donor polynucleotide comprising sequence having at least one nucleotide change relative to the target chromosomal sequence. The donor polynucleotide has substantial sequence identity to sequence at or near the targeted site in the chromosomal sequence such that the double-stranded break introduced by the targeting endonuclease can be repaired by a homology-directed repair process and the sequence of the donor polynucleotide can be inserted into or exchanged with thechromosomal sequence, thereby modifying the chromosomal sequence. For example, the donor polynucleotide can comprise a first sequence having substantial sequence identity to sequence on one side of the target site and a second sequence having substantial sequence identify to sequence on the other side of the target site. The donor polynucleotide can further comprise a donor sequence for integration into the targeted chromosomal sequence. For example, the donor sequence can be an exogenous sequence (e.g., a marker sequence) such that integration of the exogenous sequence disrupts the reading frame and inactivates the targeted chromosomal sequence.

[0087] The lengths of the first and second sequences in the donor polynucleotide that have substantial sequence identify to sequences at or near the target site in the chromosomal sequence can and will vary. In general, each of the first and second sequences in the donor polynucleotide is at least about 10 nucleotides in length. In various embodiments, the donor polynucleotide sequences having substantial sequence identify with chromosomal sequences can be about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, or more than 100 nucleotides in length.

[0088] The phrase “substantial sequence identity" means that the sequences in the polynucleotide have at least about 75% sequence identify with the chromosomal sequences of interest. In some embodiments, the sequences in the polynucleotide about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identify with the chromosomal sequences of interest.

[0089] The length of the donor polynucleotide can and will vary. For example, the donor polynucleotide can range from about 20 nucleotides in length up to about 200,000 nucleotides in length. In various embodiments, the donor polynucleotide can range from about 20 nucleotides to about 100 nucleotides in length, from about 100 nucleotides to about 1000 nucleotides in length, from about 1000 nucleotides to about 10,000 nucleotides in length, from about 10,000 nucleotides to about 100.000 nucleotides in length, or from about 100,000 nucleotides to about 200,000 nucleotides in length.

[0090] Typically, the donor polynucleotide is DNA. The DNA can be single-stranded or double-stranded. The DNA can be linear or circular. In some embodiments, the donorpolynucleotide can be an single-stranded, linear oligonucleotide comprising less than about 200 nucleotides. In other embodiments, the donor polynucleotide can be part of a vector. Suitable vectors include DNA plasm ids, viral vectors, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC). In still other embodiments, the donor polynucleotide can be a PCR fragment or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.

[0091] The donor polynucleotide(s) can be introduced into the cells at the same time as the targeting endonuclease molecule(s). Alternatively, the donor polynucleotide(s) and the targeting endonuclease molecule(s) can be introduced into the cells sequentially. The ratio of the targeting endonuclease molecule(s) to the donor polynucleotide(s) can and will vary. In general, the ratio of targeting endonuclease molecule(s) to donor polynucleotide(s) ranges from about 1: 10 to about 10:1. In various embodiments, the ratio of the targeting endonuclease molecule(s) to polynucleotide(s) can be about 1: 10, 1:9, 1:8, 1 :7, 1:6, 1:5, 1 :4, 1 :3, 1 :2, 1: 1, 2: 1. 3: 1, 4: 1, 5: 1. 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. In one embodiment, the ratio is about 1 : 1.

[0092] The method further comprises maintaining the cell under appropriate conditions such that the double-stranded break introduced by the targeting endonuclease can be repaired by(i) a non-homologous end-joining repair process such that the chromosomal sequence is modified by a deletion, insertion and / or substitution of at least one nucleotide or, optionally,(ii) a homology -directed repair process such that the chromosomal sequence is exchanged with the sequence of the polynucleotide such that the chromosomal sequence is modified. In embodiments in which nucleic acid(s) encoding the targeting endonuclease(s) is introduced into the cell, the method comprises maintaining the cell under appropriate conditions such that the cell expresses the targeting endonuclease(s).

[0093] In general, the cell is maintained under conditions appropriate for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Umov et al. (2005) Nature 435:646-651; and Lombardo et al (2007) Nat. Biotechnology 25: 1298-1306. Those of skill in the art appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routineoptimization may be used, in all cases, to determine the best techniques for a particular cell type.

[0094] During this step of the process, the targeting endonuclease(s) recognizes, binds, and creates a double-stranded break(s) at the targeted cleavage site(s) in the chromosomal sequence, and during repair of the double-stranded break(s) a deletion, insertion, and / or substitution of at least one nucleotide is introduced into the targeted chromosomal sequence. In specific embodiments, the targeted chromosomal sequence is inactivated.

[0095] Upon confirmation that the chromosomal sequence of interest has been modified, single cell clones can be isolated and genotyped (via DNA sequencing and / or protein analyses). Cells comprising one modified chromosomal sequence can undergo one or more additional rounds of targeted genome modification to modify additional chromosomal sequences, thereby creating double knock-out, triple knock-outs, and the like.

[0096] Another aspect of the present disclosure encompasses methods for producing recombinant proteins with reduced levels of residual AEP or reducing the level of AEP contamination in recombinant proteins produced in a biologic production system. The methods comprise expressing the recombinant protein of interest in any of the engineered cell lines described above and purifying the expressed recombinant protein.

[0097] Recombinant proteins produced by the engineered cell lines disclosed herein have reduced levels of AEP as compared to recombinant proteins produced by the non-engineered parental cell lines. In general, the residual levels of AEP in recombinant proteins produced by the cell lines disclosed herein are less than 100 ppm, less than 30 ppm, less than 10 ppm, less than 3 ppm, less than 1 ppm, less than 0.3 ppm, less than 0.1 ppm, less than 0.03 ppm, less than 0.01 ppm, less than 0.003, or less than 0.001 ppm, as measured using validated methods in accordance with International Conference on Harmonization (ICG) guidelines. Suitable methods include Western immunoblotting assays, ELISA enzyme assays, one- or two- dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE), 2D-differential in-gel electrophoresis (DIGE), capillary' zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS / MS), liquid chromatography -tandem mass spectrometry (LC- MS / MS), two-dimensional-liquid chromatography-tandem mass spectrometry (2D-LC- MS / MS), and the like.Culture Methods

[0098] Various culture methods may be used to produce a recombinant protein of interest, including, but not limited to, batch culture, fed-batch culture, perfusion culture, and intensified cell culture.

[0099] Batch culture is a discontinuous method where cells are grown in a fixed volume of culture media for a short period of time followed by a full harvest. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, followed by a decline in viable cell density as the media components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Harvest typically occurs at the point when the maximum cell density is achieved (e.g., 5 x 106cells / mL or greater, depending on media formulation, cell line, etc.). The batch process is the simplest culture method; however, viable cell density is limited by nutrient availability and once the cells are at maximum density, the culture declines and production decreases. There is no ability to extend a production phase in batch culture because the accumulation of waste products and nutrient depletion rapidly lead to culture decline, typically around 3 to 7 days.

[0100] Fed-batch culture improves on the batch process by providing bolus or continuous media feeds to replenish those media components that have been consumed. Since fed-batch cultures receive additional nutrients throughout the run, they have the potential to achieve higher cell densities (>10 to 30xl06cells / mL, depending on media formulation, cell line, etc.) and increased product titers, when compared to the batch method. Unlike the batch process, a biphasic culture can be created and sustained by manipulating feeding strategies and media formulations to distinguish the period of cell proliferation to achieve a desired cell density (the grow th phase) from the period of suspended or slow cell growth (the production phase). As such, fed-batch cultures have the potential to achieve higher product titers compared to batch cultures. Typically, a batch method is used during the growth phase and a fed-batch method used during the production phase, but a fed-batch feeding strategy can be used throughout the entire process. However, unlike the batch process, bioreactor volume is a limiting factor which limits the amount of feed. Also, as with the batch method, metabolic by-product accumulation will lead to culture decline, which limits the duration of the production phase, often around 10 to 21 days. Fed-batch cultures are discontinuous, and harvest typically occurs when metabolic by-product levels or culture viability reachpredetermined levels. When compared to a batch culture, in which no feeding occurs, a fed-batch culture can produce greater amounts of recombinant protein. (See, e.g.. U.S. Patent No. 5,672,502.)

[0101] Perfusion methods offer potential improvements over the batch and fed-batch methods by adding fresh media and simultaneously removing spent media during culture. Typical perfusion cultures begin with a batch culture start-up lasting for a day or two followed by continuous, step-wise, and / or intermittent addition of fresh feed media to the culture and simultaneous removal of spent media with the retention of cells and additional high molecular weight compounds such as proteins (based on the fdter molecular weight cutoff) throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent media, while maintaining cell density. Non-limiting examplse of filtration methods include alternating tangential flow filtration and recirculating tangential flow. Alternating tangential flow is maintained by pumping medium through hollow-fiber filter modules. See e.g. US Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22 (7):62-63.

[0102] Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of any of these. Perfusion rates can be less than a working volume to many working volumes per day. The cells are retained in the culture, and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture.

[0103] Typical large scale commercial cell culture strategies strive to reach high cell densities, such as, e.g., 30 - 90(+) x 106cells / mL, where almost a third to over one-half of the reactor volume is biomass. With perfusion culture, extreme cell densities of >1 x 108cells / mL have been achieved. A potential advantage of the perfusion process is that the production culture can be maintained for longer periods than batch or fed-batch culture methods. However, increased media preparation, use, storage, and disposal are necessary to support a long-term perfusion culture, particularly for a culture with high cell density’, which also needs even more nutrients. All of this can increase production costs compared to batch and fed-batch methods. In addition, higher cell densities can cause problems during production, such as, e.g., maintaining dissolved oxygen levels and problems with increased gassing, including supplying more oxygen and removing more carbon dioxide, which couldresult in more foaming and the need for alterations to antifoam strategies; as well as during harvest and downstream processing where the efforts required to remove the excessive cell material can result in loss of product, negating the benefit of increased titer due to increased cell mass.

[0104] Suitable culture conditions, including temperature, dissolved oxygen content, agitation rate, and the like, for mammalian cells are known in the art and may vary by the phase or stage of the cell culture. In some embodiments, the methods disclosed herein further comprise taking samples during the cell culture processes, evaluating the samples to quantitatively and / or qualitatively monitor characteristics of the recombinant protein and / or the cell culture process. In some embodiments, the samples are quantitatively and / or qualitatively monitored using process analytical techniques. For examples, dissolved oxygen levels may be monitored during the cell culture processes using methods known in the art, such as, e.g., a basic chemical analysis method (titration method), an electrochemical analysis method (diaphragm electrode method), and a photochemical analysis method (fluorescence method).

[0105] During recombinant protein production, it is desirable to have a controlled system where cells are grown for a desired time or to a desired density and then the physiological state of the cells is switched to a growth-limited or arrested, high productivity state where the cells use energy and substrates to produce the recombinant protein in favor of increasing cell density. For commercial scale cell culture and the manufacture of biological therapeutics, the ability to limit or arrest cell growth and being able to maintain the cells in a growth-limited or arrested state during the production phase is very desirable. Such methods include, for example, temperature shifts, use of chemical inducers of protein production, nutrient limitation or starvation and cell cycle inhibitors, either alone or in combination. Illustratively, a typical cell culture undergoes a growth phase, this is a period of exponential growth where cell density is increased. During the growth phase, cells are cultured in a cell culture medium containing the necessary nutrients and additives under conditions (generally at about a temperature of 25°-40°C, in a humidified, controlled atmosphere) such that optimal growth is achieved for the particular cell line. Cells are typically maintained in the growth phase for a period of between one and eight days, e.g., between three to seven days, e.g., seven days. The length of the growth phase for a particular cell line can be determined by a person of ordinary skill in the art and will generally be the period of time sufficient to allow the particular cellsto reproduce to a viable cell density within a range of about 20% -80% of the maximal possible viable cell density if the culture was maintained under the growth conditions. The growth phase is followed by a transition phase when exponential cell growth is slowing and protein production starts to increase. This marks the start of the stationary phase, a production phase, where cell density typically levels off and product titer increases. During the production phase, the medium is generally supplemented to support continued recombinant protein production.

[0106] In certain embodiments of the methods of the present disclosure, the culture conditions may be adjusted to facilitate the transition from the grow th phase of the cell culture to the production phase. For instance, a growth phase of the cell culture may occur at a higher temperature than a production phase of the cell culture. In some embodiments, a growth phase may occur at a first temperature from about 35°C to about 38°C, and a production phase may occur at a second temperature from about 29°C to about 37°C, optionally from about 30°C to about 36°C or from about 30°C to about 34°C. In one embodiment, a shift in temperature from about 35°C to about 37°C to a temperature of about 31 °C to about 33°C may be employed to facilitate the transition from the growth phase of the culture to the production phase. Chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time as, before, and / or after a temperature shift, or in place of a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.

[0107] Additionally, any cell culture media capable of supporting growth of the appropriate host cell in culture can be used. Typically, the cell culture medium contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability', and / or recombinant protein production in a particular cultured host cell, are commercially available and include RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™ 300 Series, among others, which can be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell culture media can be serum-free, protein-free, growth factor-free, and / orpeptone-free media. Cell culture media may also be enriched by the addition of nutrients or other supplements, which may be used at greater than usual, recommended concentrations. In certain embodiments, the culture medium used in the methods of the present disclosure is a chemically defined medium, which refers to a cell culture medium in which all of the components have known chemical structures and concentrations. Chemically defined media are typically serum-free and do not contain hydrolysates or animal-derived components.

[0108] Various media formulations can be used during the life of the culture, for example, to facilitate the transition from one stage (e.g., the growth stage or phase) to another (e.g., the production stage or phase) and / or to optimize conditions during cell culture (e.g. concentrated media provided during a perfusion culture). A growth medium formulation can be used to promote cell growth and minimize protein expression. A production medium formulation can be used to promote production of the recombinant protein of interest and maintenance of the cells, with minimal new cell growth. A feed medium is typically a cell culture medium containing more concentrated components such as nutrients and amino acids, which are consumed during the course of the production phase of the cell culture. Feed medium may be used to supplement and maintain an active culture, particularly a culture operated in fed batch, semi-perfusion, or perfusion mode. Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5x, 6x, 7x, 8x, 9x, 10x. 12X, 14x, 16x. 20x, 30x, 50x. 10()x, 200 , 400x. 600x, 800 , or even about 1000x of their normal amount.

[0109] In some embodiments of the methods of the present disclosure, the mammalian cell is cultured for a defined period of time during which the recombinant protein is expressed and secreted by the mammalian cell. This period of time (i.e. the duration of the production phase of the cell culture) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the production phase of the cell culture is about 7 days to about 28 days, about 10 days to about 30 days, about 7 days to about 14 days, about 10 days to about 18 days, about 3 days to about 15 days, about 5 days to about 8 days, about 12 days to about 15 days, about 12 days to about 18 days, or about 15 days to about 21 days. In some embodiments, the duration of the production phase of the cell culture is 7 days, 8 days, 9 days, 12 days, 15 days, 18 days, or 21 days.

[0110] In some embodiments of the methods of the present disclosure, the biomanufacturing process comprises a production phase with a viable cell density of at least 100 x 105cells / mL, for example between about 100 x 105cells / mL and about 10 x 107cells / mL, between about 250 x 105cells / mL and about 900 x 105cells / mL, between about 300 x 105cells / mL and 800 x 105cells / mL, or between about 450 x 105cells / mL and 650 x 105cells / mL. Cell density may be measured using a hemacytometer, a Coulter counter, or an automated cell analyzer (e.g. Cedex automated cell counter). Viable cell density may be determined by staining a culture sample with Trypan blue, which is taken up only by dead cells. Viable cell density is then determined by counting the total number of cells, dividing the number of stained cells by the total number of cells, and taking the reciprocal.[OHl] In some embodiments of the methods of the present disclosure, the biomanufacturing process comprises a production phase with a packed cell volume less than or equal to 35%. In some embodiments, the packed cell volume is less than or equal to 30%.Purification Processes

[0112] The expressed recombinant proteins may be secreted into the culture medium from which they can be recovered and / or collected. Harvest operations comprising an acid precipitation may be combined with additional harvest strategies, including centrifugation, such as disk-stack centrifugation, intermittent discharge centrifugation, or continuous solid discharge centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration, and depth filtration; precipitation / sedimentation methods, such as flocculation; and chromatography media-based separations.

[0113] In general, the recombinantly produced protein is secreted into culture medium, so harvesting typically involves a step of harvesting the supernatant, wherein cells, cell debris, or other large particulates are separated from the recombinant protein. Such operations may include cooling, flocculation, acidification, centrifugation, neutralization, acoustic wave separation, and various forms of filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow- filtration). The harvested cell culture fluid may be further clarified to remove fine particulate matter and soluble aggregates by filtration with a membrane having a pore size between about 0. 1 pm and about 0.5 pm, such as, e.g.. a membrane having a pore size of about 0.22 pm.

[0114] Beyond a harvest operation comprising an acid precipitation, the present disclosure comprehends methods involving all known post-harvest recovery technologies, such as, e.g.. protein A purification of immunoglobulin and immunoglobulin-like biologies, as well as chromatography-based separations and polishing steps that include column and alternative modes of chromatographic separations by ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX). hydrophobic interaction chromatography (HIC). mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reverse-phase chromatography, size exclusion chromatography (SEC), gel filtration, or any other know n form of chromatographic separation of biological and / or biochemical substances.

[0115] In some embodiments of the methods of the present disclosure, recombinant protein recovered from the host cells or cell culture medium may be further purified or partially purified to remove cell culture media components, host cell proteins, or nucleic acids, or other process or product-related impurities by one or more unit operations. One of ordinaryskill in the art can select the appropriate unit operation(s) for further purification of a recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of host cell from which the recombinant protein is expressed, and the composition of the culture medium in which the host cells were grown. Illustratively, in some embodiments, the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0116] A capture unit operation may include capture chromatography that makes use of resins and / or membranes containing agents that will bind to the recombinant protein of interest, for example, affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like. Such chromatographic materials are known in the art and are commercially available. For instance, if the recombinant protein is an antibody or contains components derived from an antibody (e.g., a Fc domain), affinity chromatography using ligands such as Protein A. Protein G, Protein A / G, or Protein L may be employed as a capture chromatography unit operation to further purify the recombinant protein. In otherembodiments, the recombinant protein of interest may comprise a polyhistidine tag at its amino or carboxyl terminus and subsequently purified using IMAC. Recombinant proteins can be engineered to include other purification tags, such as a FLAG® tag or c-myc epitope and subsequently purified by affinity chromatography using a specific antibody directed to such tag or epitope.

[0117] Additional unit operations to inactivate, reduce, and / or eliminate viral contaminants may include filtration processes and / or adjusting solution conditions. One method for achieving viral inactivation is incubation at low pH (e.g., pH<4). A low pH viral inactivation operation can be followed with a neutralization unit operation that readjusts the virus inactivated solution to a pH more compatible with the requirements of the subsequent unit operations. A low pH viral inactivation operation may also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitation. Adjusting the temperature or chemical composition (e.g., use of detergents) can also be used to achieve viral inactivation. Viral filtration can be performed using micro- or nano-filters, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0118] A polishing unit operation may make use of various chromatographic methods for the purification of the protein of interest and clearance of contaminants and impurities. The polish chromatography unit operation may make use of resins and / or membranes containing agents that can be used in either a ’‘flow-through mode,’’ in w hich the protein of interest is contained in the eluent and the contaminants and impurities are bound to the chromatographic medium, or “bind and elute mode,” in which the protein of interest is bound to the chromatographic medium and eluted after the contaminants and impurities have flowed through or been washed off the chromatographic medium. Examples of such polish chromatography methods include, but are not limited to, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reverse phase chromatography, and size-exclusion chromatography (e.g. gel filtration).

[0119] Purified recombinant protein may be formulated, i.e., buffer exchanged, sterilized, bulk-packaged, and / or packaged for a final user. Illustratively, product concentration and buffer exchange of the recombinant protein of interest into a desired formulation buffer forbulk storage of the drug substance or drug product can be accomplished by ultrafiltration and diafiltration. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.Recombinant Proteins

[0120] Any type of recombinant protein comprising an AEP cleavage site, including proteins containing single polypeptide chains or multiple polypeptide chains, can be harvested according to the methods of the present disclosure. Recombinant proteins of the present disclosure include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Illustratively, recombinant proteins can include, but are not limited to, cytokines, grow th factors, hormones, muteins, fusion proteins, antibodies, antibody fragments, peptibodies, T-cell engaging molecules, and multi-specific antigen binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0121] In other embodiments, the recombinant protein to be harvested according to a method of the present disclosure is an antigen-binding protein. Antigen-binding proteins include, but are not limited to, antibodies, peptibodies. antibody derivatives, antibody analogs, fusion proteins (including, e.g., single-chain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sei 23:221-228)), hetero- IgGs (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Additional antigen-binding proteins include, but are not limited to, bispecific T cell engager (BiTE®) molecules, bispecific T cell engagers having extensions, such as, e.g., half-life extensions, such as, e.g., HLE BiTE molecules, Heterolg BITE molecules, and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).

[0122] In some embodiments, the antigen-binding protein binds to one of more of the following, alone or in any combination: CD proteins including, but not limited to, CD3, CD4, CD5, CD7. CD8, CD19, CD20, CD22, CD25. CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138. CD171, and CD174. HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor, EGFRvIII, cell adhesion molecules, for example, LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin, grow th factors, including but not limited to, for example, vascular endothelial growth factor(“VEGF ’); VEGFR2, grow th hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto. transforming growth factors (TGF), including, among others. TGF-a and TGF-0. including TGF-01. TGF-02. TGF-03. TGF-04, or TGF-05. insulin-like growth factors-I and -II (IGF -I and IGF-II), des(l-3)-IGF-I (brain IGF -I), and osteoinductive factors, insulins and insulin-related proteins, including, but not limited to, insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor, colony stimulating factors (CSFs), including the following, among others, M-CSF. GM-CSF, and G-CSF. other blood and serum proteins, including but not limited to albumin. IgE, and blood group antigens, receptors and receptor-associated proteins, including, for example, Hk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptors, and T-cell receptors; neurotrophic factors, including but not limited to, bone- derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4. NT-5, or NT-6); relaxin A-chain, relaxin B-chain, and prorelaxin, interferons, including for example, interferon-alpha, -beta, and -gamma, interleukins (ILs), e.g., IL-1 to IL-10, IL-12, IL-15, IL- 17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 to the receptor, IL-13RA2, or IL-17 receptor, IL-1RAP; viral antigens, including but not limited to, an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF). AIDS envelope, transport proteins, homing receptors, MIC (MIC -a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, antigen-binding proteins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA. LMP1. MG7, NY-ESO-1, PSCA, ganglioside GD2. ganglioside GM2, BAFF, OPGL(RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit. B7RP-1, PSMA. NKG2D-1. programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGP, hepatitis-C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (lly), IFN gamma, interferon gamma induced protein 10 (IP 10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin Type 9 (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP). OX40L, a4P7, platelet specific (platelet glycoprotein Ilb / IIIb (PAC-1). transforming growth factor beta (TFGP), Zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0123] In other embodiments, the recombinant protein to be harvested according to a method of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody.

[0124] In some embodiments, the antibody is selected from abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, ontamalimab, ocrelizumab, panitumumab, prezalumab, ravulizumab, rilotumumab. romosozumab. satralizumab, tafolecimab. tanezumab, tezepelumab, tremelimumab, utomilumab, and volagidemab. In some embodiments, the antibody is selected from denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody is erenumab. In some embodiments, the antibody is evolocumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is tezepelumab. In some embodiments, the antibody is ocrelizumab.

[0125] In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is a human IgGl antibody.Monitoring AEP Quantity or Activity or LMW Species

[0126] Some embodiments of certain methods of the present disclosure involve monitoring AEP quantity or activity and / or monitoring a quantity of LMW species of a desired recombinant protein.

[0127] In some embodiments, AEP quantity is measured using mass spectrometry or an immunoassay. In some embodiments, AEP quantity is measured using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, AEP quantity is measured using nano-scale liquid chromatography-mass spectrometry (nano LC-MS).

[0128] In some embodiments, AEP activity is measured using a cleavage assay. In some embodiments in which the biomanufacturing process utilizes CHO cells, AEP peptides containing the activating auto-cleavage sites N325, D3(and E311(LMSTN325DLK) (SEQ ID NO:32) and LD305LTPSPE311VPLTILK) (SEQ ID NO:33) may be used as indicators of cleavage and activation. AEP peptides containing alternative auto-cleavage sites may be used to monitor activation for biomanufacturing processes utilizing alternative mammalian host cells.

[0129] In some embodiments, AEP proenzyme quantity is measured using mass spectrometry or an immunoassay. In some embodiments, AEP proenzyme quantity is measured using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, AEP proenzyme quantity is measured using nano-scale liquid chromatography -mass spectrometry (nano LC- MS). In some embodiments in which the biomanufacturing process utilizes CHO cells, AEP peptides containing the activating auto-cleavage sites N325, D305and E311(LMSTN325DLK and LD305LTSPE311VPLTILK) (SEQ ID NO:32 and SEQ ID NO:33, respectively) are used as indicators of cleavage and activation from AEP proenzyme to mature AEP.

[0130] In some embodiments, the quantity of LMW species is measured using mass spectrometry or an immunoassay, e.g., a bioassay and / or potency assay. In some embodiments, the quantity of LMW species is measured using a reduced capillary electrophoresis-sodium dodecyl sulfate method. In some embodiments, the quantity of LMW species is measured using a reduced reversed-phase (RP) chromatography method. In some embodiments, the quantity of LMW species is measured using an ultra-high-pressure liquid chromatography method.Definitions

[0131] In some embodiments, “about,” when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95%confidence interval for the mean) or ± 10% of the indicated value, whichever is greater. In some embodiments, numeric ranges are inclusive of the numbers defining the range (i.e.. the endpoints).

[0132] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0133] The terms “a” and "an" mean “one or more’' unless specifically indicated otherwise. Additionally, “one or more” and “at least one” are used interchangeably herein. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0134] The term “acid precipitation” refers to a harvest operation in which cell culture pH is reduced to induce precipitation of one or more cell culture impurities.

[0135] The term “cell culture” or “culture” refers to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian and bacterial cells are known in the art. (See, e.g, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992).) Mammalian cells may be cultured in suspension or while attached to a solid substrate. In some embodiments, fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, and / or stirred tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0136] The term “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of live cells in a given volume of culture medium, as determined by standard viability assays (such as, e.g., a trypan blue dye exclusionmethod) and may be measured at any point during a specific phase of a cell culture process. As used herein, the term “packed cell volume’" (PCV), also referred to as “percent packed cell volume” (%PCV), is the ratio of the volume occupied by the cells, to the total volume of cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6): 1228-33). Packed cell volume is a function of cell density and cell diameter; increases in packed cell volume could arise from increases in cell density or cell diameter or both. Packed cell volume is a measure of the solid content in the cell culture. Since host cells vary in size and cell cultures also contain dead and dying cells and other cellular debris, packed cell volume can describe with a greater degree of accuracy the solid content within a cell culture.

[0137] A “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. A host cell that comprises a nucleic acid encoding a recombinant protein, e.g., operably linked to at least one expression control sequence (e.g. promoter or enhancer), is a “recombinant host cell.” A host cell, when cultured under appropriate conditions, may synthesize a recombinant protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).

[0138] “Low molecular weight” or "LMW" species of a recombinant protein of interest refer to fragments, truncated forms, or other incomplete variants of the recombinant protein that have a molecular weight less than the molecular weight of the intact, fully assembled form of the recombinant protein. LMW species can include, but are not limited to, proteolytic fragments, truncated forms resulting from cellular expression of mRNA splice variants, and single component polypeptides in the case of multi-polypeptide chain proteins (e.g. light chain or heavy chain only species when the recombinant protein is an antibody).

[0139] The term “protease inhibitor” refers to a molecule that at least partially inhibits the function of one or more protein-based enzymes that cleave other proteins. In some embodiments of the present disclosure, an exogenous protease inhibitor is added to a cell culture to inhibit cleavage of the recombinant protein of interest. In some embodiments, the exogenous protease inhibitor is a commercially available protease inhibitor cocktail intendedto increase secreted protein stability, such as, e.g., E-64 protease inhibitor, TCM ProteaseArrest™ Protease Inhibitor Cocktail (G-Biosciences), Protease Inhibitor Cocktail I (R&D Systems), Halt™ Protease Inhibitor Cocktail (Thermo Scientific), or Protease Inhibitor Cocktail (Promega or Sigma- Aldrich). In some embodiments, the exogenous protease inhibitor is an AEP inhibitor. In some embodiments, the AEP inhibitor is AENK, which is commercially available, e.g., from Sigma- Aldrich. In some embodiments, the AEP inhibitor is 5-secretase inhibitor 11 (7-morpholin-4-yl-benzo[I.2.5]oxadiazol-4-ylamine). In some embodiments, the AEP inhibitor is 7-morpholinobenzo[c][l,2,5]oxadiazol-4-amine. In some embodiments, the AEP inhibitor is a substituted 3, 7-dihy dropurine-2, 6-dione derivative described in U.S. Patent Application Publication No. 2017 / 0166569.

[0140] The term “knockout”, as related to genomics, refers to an alteration in the nucleic acid sequence of a gene that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% compared to the unaltered gene. The alteration, for example, may be an insertion, substitution, deletion, frameshift mutation, or missense mutation.

[0141] The term "knockdown" refers to techniques by which the expression of one or more genes is reduced, either through genetic modification (a change in the DNA of one of the organism's chromosomes) or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene. If genetic modification of DNA is done, the result is a "knockdown organism" or "knockdown host cell".EXAMPLES

[0142] In order that the present disclosure may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosure in any manner.Summary

[0143] A low level of fragmentation was detected by rCE-SDS during the development of a high yield biomanufacturing process in CHO cells for an IgGl antibody mAbl (Figure 1). Reversed-phase (RP) LC-UV-MS analysis determined that fragmentation was occurring at a - NL- cleavage site (wherein the peptidyl bond between N and L was cleaved); this site is arelatively conservative motif in the CDR region in light chain of monoclonal antibodies, and the motif occurs in several other antibodies.

[0144] Nano LC-MS proteomics identified the presence of enzyme asparaginyl endopeptidase (AEP), also known as legumain (LGMN), in biomanufacturing process pools. AEP is a lysosomal cysteine endopeptidase, which exhibits fragmentation activity at mildly acidic pH (2.5 < pH < 4.5) and negligible activity at pH 5 and above (Zhao, L. et al.. Cell Res 2014, 24 (3), 344-358). AEP is originally present during cell culture in a proenzyme form, which converts to mature, active form by auto cleavage of terminal domains at low pH. The studies described below confirmed the presence of AEP in the high yield biomanufacturing process and revealed that AEP was cleaving mAbl primarily at low pH during an acid precipitation operation of antibody harvest from the bioreactor. Acid precipitation is a step used to precipitate cells, DNA, and other cell debris before downstream purification, while maintaining an antibody in a soluble form. Nano LC-MS analysis also showed that a portion of the AEP proenzyme form was converted to a mature, active form by auto-cleavage of the C-terminal cap domain at low pH during acid precipitation. Because AEP enzymatic activity is higher at low pH, raising the mAb incubation pH from 4.0 to 5.2 led to a decrease in fragmentation, as demonstrated by RP LC-UV-MS. Addition of AEP inhibitor AENK decreased fragmentation even at pH 4.0. A second, genetic knockout (KO) study was also performed in which the AEP gene was removed from mAbl expression cell lines, and the formation of -N^L- cleavage fragments were prevented at both pH 4.6 and pH 4.0. Taken together, this work identified AEP cleavage as a cause of fragmentation during low pH acid precipitation operations for recombinant proteins comprising an AEP cleavage site, such as - NL- motif, and provided a mechanistic based understanding and mitigation strategy.Example 1. Materials and Methods

[0145] In-process samples of mAbl and final drug substance (DS) of mAbl samples were collected from supernatant before acid precipitation and harvested cell culture fluid (HCCF) after acid precipitation stages from downstream purification process (FIGs. 2A-2B). A nano LC-MS based proteomics approach was used to identify and quantify AEP levels relative to the mAb product for the samples (FIGs. 3A-3D: FIGs. 8A-8C, Table 2). Both the mature and proenzyme forms of AEP were measured and correlated with mAbl fragmentation levels at different stages of the process. mAbl fragment products were identified and quantified byreduced RPLC-UV-MS, and rCE-SDS-UV methods. For further confirmation, commercially available AEP inhibitor AENK was spiked into mAb samples at different levels (FIG. 4; FIG. 5B), which showed decrease in fragmentation in inhibitor dose-dependent manner (FIGs. 5A, 6A-6B, 7A-7B). Higher level of the inhibitor resulted in greater decrease of the fragmentation. Additionally, a genetic knockout study was performed in which mAbl was expressed in cells with deleted gene for AEP, and the -N^L- fragmentation was eliminated.1.1 Sample Preparation for analytical methods.

[0146] Trypsin Digest for Nano LC-MS Proteomics. Trypsin Digest for Nano LC-MS Proteomics. 500 pg of each mAbl sample were taken and placed into 10K MWCO Amicon centrifugal filter tubes. Yeast standard proteins glucose-6-phosphate dehydrogenase (G6PD) and inorganic pyrophosphatase (IPYR) were spiked into each sample at 100 and 20 ng / mg, respectively, and all samples were spun for 30 minutes at 14,000 rpm using a Beckman Coulter microfuge 18 centrifuge. 400 pL of denaturing buffer (6M Guanidine-HCl / pH 7.5 / 2mM EDTA / 20mM Methionine / 250mM Tris) and 6 pL 0.5 M 1,4-Dithiothreitol (DTT) were added to the samples, which were then incubated at 37 °C for 30 minutes. Samples were then alkylated in dark conditions for 30 minutes by adding 14 pL of 0.5M iodoacetic acid (1AA). Alkylation was quenched by adding 8 pL 0.5M DTT. After quenching, the denaturing buffer was spun through the 10K MWCO Amicon centrifugal filter at 14,000 rpm for 30 minutes. The flow-through was discarded and 400 pL 0.1M TRIS at pH 7.5 were added before spinning for another 30 minutes at 14,000 rpm. This step was repeated one more time. 25 pL of trypsin solution aliquots (1 pg / mL) were then added to the samples, followed by the addition of 50 pl 0.1 M TrisHCl at pH 7.5. After the samples were gently mixed, they were placed in an incubator at 37°C for overnight digestion (18-20 hours). 100 pL 7.5 M Guanidine-HCl at pH 5.0 was added into samples, which were then gently shaken for 30 minutes. All samples were spun for 45 minutes at 14,000 rpm using a Beckman Coulter microfuge 18 centrifuge to collect flow-through (about 200 pl) for LC / MS analysis.

[0147] Acid precipitation. For confirmation of loss of AEP activity after genetic knockout, the day 15 supernatant samples were subjected to acid precipitation by titration with 1 M citric acid. Two aliquots of each harvest supernatant were titrated to pH 4.6 and pH 4.0 followed by incubation at room temperature for up to 48 hrs. Samples were taken from theacid precipitation aliquots at various time intervals for analysis by reduced RPLC-UV-MS to monitor for AEP cleavage products.

[0148] Disulfide Reduction for reduced RPLC-UV-MS of mAbl. Supernatant samples were prepared for reduced LC-MS intact mass analysis by denaturation in 6 M guanidine hydrochloride with 250 mM TRIS buffer and 20 mM DTT at pH 7.8 for 30 minutes at 37 °C.1.2 Analytical Methods.

[0149] Nano LC for proteomics. A nanoLC system (Easy-nLC 1200, Thermo Fisher Scientific) was used to separate the tryptic peptides that were collected after sample preparation. An analytical nanoflow column (75pm x 250 mm, EASY-Spray™ HPLC from Thermo Scientific) with a particle size of 2 pm and a trap column (75pm x 20 mm, Acclaim™ PepMap™ 100 C18 HPLC Column from Thermo Fisher Scientific) were used, with the trap column in front of the analytical column. The peptide digests were first loaded into the trap column and then eluted from there to the analytical column for further separation. Mobile phase A was 0.1% formic acid / 99.9% water and mobile phase B was 0.1% formic acid / 19.9% water / 80% acetonitrile. The flow rate was 300 nL / min, with the column temperature set at 40 °C. A gradient of 3% to 40% B over 80 minutes was used.

[0150] MS for proteomics . A Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) was coupled with the nanoLC system for the LC-MS analysis. The nano flow source conditions were set up as follows: spray voltage=1.9 KV, transfer capillary temperature=325 C°, RF lens =50%. The full MS scans were acquired using the profile data mode with resolution of 120,000 AGC target of 300%, Maximum injection time of 25 ms, and m / z scan range from 350-1400. The MS / MS scans were acquired using the centroid data with resolution of 17,500. AGC target of 50%, and a maximum injection time of 100 ms. The top ten most abundant peptide ions were selected for MS / MS with dynamic exclusion for 30 seconds.

[0151] Reduced capillary electrophoresis with SDS (rCE-SDS) of mAbl. The reduced and denatured protein samples were electrokinetically injected into a bare fused silica capillary (50 pm ID x 30.0 cm effective length), separated using SDS gel buffer, followed by UV detection at 220 nm by a photodiode array detector on a ProteomeLab PA800 PLUS CE system (Beckman Coulter, Brea, CA).

[0152] Reversed-phase LC-UV-MS of mAbl. Agilent 1290 II HPLC system was used to separate light and heavy chains and fragments of the reduced mAh. Mobile phase A was 0.1% FA / 99.9% water and mobile phase B was 0.1% FA / 89.9% propanol. An analytical regular column (2. 1 mm x 50 mm, Zorbax SB C8, Agilent) was used with flow rate of 500 ul / min under temperature of 75C. A gradient of 22% to 30% B over 5 minutes was used to elute the chains and fragments. The HPLC was connected online with the Exploris 480 mass spectrometer for analysis.

[0153] Data Processing for proteomics . A MassAnalyzer 6.03 was used to process the MS data and generate a MGF file for Mascot database search. The MS noise level was set at 1,300,000 with a minimal signal to noise ratio of 5.

[0154] Mascot Database Search for proteomics HCP Identification. After the searchable file (MGF file) was generated by MassAnalyzer, it was used for Mascot database search for Host Cell Protein (HCP) identification. The search parameters used are listed below: Peptide Tolerance: 10 ppm, MS / MS Tolerance: 10 ppm or less, Primary Digest Reagent: Trypsin; Secondary Digest Reagent: None; Missed Cleavages:0; Fixed Modifier Reagents: Carbamidomethyl C; Database UniProt CHO 2013.

[0155] HCP Quantification using Proteomics Data. MassAnalyzer used the average peak area of the top three most abundant peptides to represent the abundance or level of the protein from which the top 3 peptides came from. The HCP level was calculated by the following formula using yeast protein Glucose-6-phosphate Dehydrogenase (G6PDH) as an internal calibrant added to the samples before digestion:HCP level (ng / mg) = the average peak area of the top 3 peptide of ((HCP area x HCP molecular weight) / (average peak area of the top 3 peptide of G6PDH x G6PDH molecular weight) x 100 ng / mg)

[0156] Reduced Intact Mass for LC Fragment Quantification. Protein Metrics By os v5.7.45 software was used for deconvolution and quantitation of reduced intact mass data to characterize AEP induced LC fragmentation of mAbl. M / z range 1500 - 7000 was used for mass deconvolution, allowing possible charges of z=2 - z=60 and a mass error of ± 5 Da for sequence based peak assignment.1.3 AEP Knockout Cell Line Generation

[0157] Cells and Cell Culture. Cell line development was commenced using pre-master cell bank (PMCB) culture of mAbl, that was used in this study to generate clones with a knockout of the asparaginyl endopeptidase (AEP), also known as legumain (LMGN), endogenous host protease.

[0158] Cell lines were maintained by passaging multiple times a week at target seed density per standard cell culture passaging method in appropriate chemically defined growth medium at 36°C, 5% CO2, and 85% humidity. Cultures were either shaken at 225 rpm with 50 mm orbital diameter in a large-capacity ISF4-X incubator (Kuhner AG, Basel, Switzerland) or incubated in a static HERAcell VIOS 160i incubator (Thermo Fisher. Waltham, MA). Culture vessels included 96-well or 24-well microtiter static plates (Coming. Coming, NY), 24-deep well micro static shaking plates (Coming, Coming, NY), 50-mL spin tubes (TPP, Trasadingen, Switzerland). Viable cell density and viability were measured with the Vi-cell BLU cell counter (Beckman Coulter, Brea, CA).

[0159] AEP / LGMN Knockout Generation. Single-guide RNAs (sgRNAs) targeting exon 1 of the asparaginyl endopeptidase (Aid’), also known as legumain (LGMN) gene from Cricetelus griseus (XM_027419160) was designed using the publicly available Synthego CRISPR Design Tool with manual adjustments to ensure the gap between the two sgRNAs is exactly 20. A combination of three different guide RNA pairs were tested. The sgRNA, rvsgmal and rvsgma3 have the sequence 5’- GAGCACCAGCACCCAGTGCC AGG-3’ (SEQ ID NO: 34) and 5’ - GGATGCTGGCAAGCACTGGG TGG - 3’ (SEQ ID NO:35) respectively. The sgRNA, referred to as rvsgma2, has the sequence 5‘- CAGTGCTTGCCAGCATCCTC AGG- 3?(SEQ ID NO:36), and the sgRNA, referred to as rvsgma4, has the sequence 5’- CCAATGGCTGGTATAATTAC CGG-3?(SEQ ID NO:37) Another sgRNA, rvsgma5, 5’- TGAGGATGCTGGCAAGCACT - 3’ (SEQ ID NO:38) was used in combination with rvsgmal. The three pairs were tested for their ability to afford clean breaks in the exon 1 region AEP gene. Synthetic sgRNA (Synthego, Redwood City, CA) was ordered with chemical modifications (2'-O-methyl at first and last bases, 3' phosphorothioate bonds between first 3 and last 2 base) introduced to the sgRNA backbone to increase the half-life of the RNA in the cell.

[0160] Purified Cas-CLOVER mRNA (Demeetra AgBio, Lexington, KY) encoded aClostridium Clo51 domain fused to the C-terminus of catalytically inactive Streptococcuspyogenes Cas9 (dCas9) with an N- terminal nuclear localization signal(NLS).OCas CLOVER transfections were performed with the 4D Nucleofector (Lonza Cologne, GmbH, Cologne, Germany) transfection system. In short, 30 pmols of rvsgma2 and rvsgma4 was mixed with lug / ul of Cas-CLOVER mRNA at room temperature 4xl05cells from PMCB culture were washed with phosphate buffered saline (PBS) solution and centrifuged at 400xg for 5 min. The cells were resuspended in 20 uL of SF Cell Line Solution +supplement 1 solution from Amaxa SF Cell Line 4D-Nucleofector X Kit S 32 RCT (Lonza, V4XC-2032). 20uL of cell solution was mixed with solution of sgmas and Cas-CLOVER mRNA. 20uL of this reaction mix was then transferred to nucleocuvette strip in appropriate orientation in Lonza 4D-Nucleofactor system and the cells were electroporated at using DT- 134 program. 80uL of CHO growth media ABM 416-009 was added to the nucleocuvette strip wells and the reaction was allowed to incubate at room temperature for 30 minutes. The entire contents of the nucleocuvette strip were transferred to 6-well plate already fdled with 3mL of ABM 415-009 growth medium. The plates were transferred to static incubator (36°C, 85% humidity, 5% CO2)

[0161] Transfected cells were single-cell sorted 12 days after transfection using the F. Sight (Cytena GmbH, Breisgau, Germany). 96-well export plates were centrifuged at 1000 rpm for 1 minute at room temperature immediately after sort, and single-cell clonali ty and colony growth were monitored by imaging on the Cell Metric (Solentim. Wimbome, United Kingdom). 14 days after single-cell cloning, 96-well export plates were sampled for viable cell density and viability'. Clones that fit the selection criteria of viability greater than 90% were subsequently scaled up by suspension adaption in 24-deep well plates and 50-mL spin tubes.

[0162] “Evidence of growth” w as determined as the presence of colonies of cell through visual inspection of the Cell Metric images in an export w ell at 14 days post-single cell cloning.

[0163] Indel Quantification by Sanger Sequencing. For indel quantification, 25 pl cells w ere lysed with 75 pl of QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI) according to manufacturer's protocol. Reaction mixture was incubated at 65 C for 6 minutes and vortexed for 15 seconds. Lysis reaction was stopped by incubating reaction mixture at 98° C for 2 minutes. The LGMN sgRNA target region in the genome was amplified usingDreamTaq Green PCR Master Mix (Thermo Fisher, Waltham, MA), a final concentration of 100 nM of forward (5’- AGGGACAGGATTGCCCTTTG -3’) (SEQ ID NO:39) and reverse (5’- ATACTGGGTCCTGAAGGCCA -3’) (SEQ ID NO:40) primers, and 1 pL of lysed DNA. PCR was performed using the ProFlex PCR System (Thermo Fisher, Waltham, MA) with the following conditions: 95°C of initial denaturation for 3 minutes followed by 40 cycles of 95°C for 30 seconds. 60°C for 30 seconds, 72°C for 1 minute, and a final extension at 72 C for 10 minutes. Sanger sequencing results from mock transfection controls and CRISPR-Cas9 transfections were uploaded into Synthego’s online Inference of CRISPR Edits (ICE) program to calculate indel efficiency.

[0164] Production Cultivation. Small-scale 10-day fed-batch production evaluations were performed per Amgen internal protocol in either 50-mL spin tubes (TPP, Trasadingen, Switzerland) with 20-mL inoculation volume. Cells were incubated at 36°C, 5% CO2, 85% relative humidity and shaken at 225 rpm with 50 mm orbital diameter for 50-mL spin tubes in a large-capacity ISF4-X incubator (Kuhner AG. Basel, Switzerland). Cultures were inoculated at a target cell density of 1 0x105cells / mL for 50-mL spin tubes and were fed a single bolus feed on days 3, 6, and 8, 10 and 13. Proprietary production and feed media were used. pH was not controlled throughout the culture duration. In-process samples were taken from cultures on days 0, 3. 6, 8, 10, and 13 for analysis. Cell counts and viability of cultures were determined using a Vi-Cell BLU cell counter (Beckman Coulter, Brea, CA) for 50-mL spin tubes. On harvest day, cell cultures were pelleted by centrifuging at 3000 rpm for 15 minutes at room temperature, and the harvest cell culture fluid was analyzed for titer. Harvest cell culture fluid from the 50-mL spin tubes was also submitted for in vitro AEP enzy matic activity. Titers were measured by affinity Protein A high performance liquid chromatography (HPLC) (Waters, Milford, MA). Doubling time was calculated at the time of fed-batch inoculation. Integrated viable cell density (IVCD) was calculated by a trapezoidal rule for VCD versus culture time. Cell-specific productivity (qp) was calculated as final titer divided by final IVCD.

[0165] mAbl expression titer determination. Protein purification of the harvest cell culture fluid via affinity chromatography (Atoll GmbH, Weingarten, Germany) was performed before product quality analysis and to determine mAb expression titers.Example 2. AEP Induced Fragmentation of an IgGl Antibody.

[0166] During the downstream purification of an IgGl monoclonal antibody (mAbl), electrophoresis with SDS (rCE-SDS) identified a small percentage of low molecular weight (LMW) species. The percentages varied for different manufacturing processes. See Table 1 and FIG. 1.Table 1

[0167] Reversed-phase LC UV-MS analysis was performed to identify the LMW species as antibody light chain fragments resulting from clipping at -NL- cleavage site, wherein AEP cleaves the peptidyl bond between residues N and L (PSN J'LASG). Clipping after N suggested enzymatic cleavage by AEP legumain. LC-MS proteomics study was then initiated to identify and quantify HCPs in in-process samples.

[0168] LC-MS proteomics identified the presence of enzy me asparaginyl endopeptidase (AEP), also known as legumain, in biomanufacturing process pools. AEP is a lysosomal cysteine endopeptidase, which exhibits fragmentation activity at mildly acidic pH (2.5 < pH < 4.5) and negligible activity at pH 5 and above (Zhao, L. et al., Cell Res 2014, 24 (3). 344- 358). AEP is originally present during cell culture in a proenzyme form, which converts to mature, active form by auto-cleavage of terminal domains at low pH. The studies described below confirmed the presence of AEP in biomanufacturing process and revealed that AEP w as cleaving mAb primarily at low pH during an acid precipitation operation of antibody harvest from the bioreactor. Acid precipitation is a common step used to precipitate cells, DNA, and other cell debris before downstream purification, while maintaining an antibody in a soluble form. LC-MS analysis also showed that the AEP proenzyme form was converted to a mature, active form by auto-cleavage of terminal portions at low pH during acid precipitation.

[0169] FIGs. 2A-2B showed two different purification process. Process in FIG. 2A does not comprise an acid precipitation step; whereas process in FIG. 2B comprises an acid purification step right after harvesting the supernatant of the cell culture. During the downstream purification with an early acid precipitation step (FIG. 2B), the in-process material with a relatively high level of HCPs is exposed to low pH activating AEP. During the alternative process where the in-process material is exposed to low pH only during Protein A elution (FIG. 2A). when HCP level is significantly (2-3 orders of magnitude) lower, than during the acid precipitation step. See, FIGs. 2C-2D.

[0170] Two IgGl mAbl in-process samples were assessed by LC-MS proteomics method in this study: 1) supernatant from bioreactor collected prior to acid precipitation step (pH 4.6) and 2) harvested cell culture fluid (HCCF) collected after acid precipitation step. Approximately 800 HCPs were identified and quantified in the samples. It was discovered that total HCP signal intensity in HCCF was decreased relative to supernatant after cell debris and HCPs were precipitated (FIG. 3A). AEP legumain intensity (quantified by top 3 peptides) remained consistent between the two samples, indicating it was not removed by acid precipitation step (FIG. 3B).

[0171] AEP pro-enzyme is activated by autocleavage of several peptides including 307- LD / LTPSPE / VPLTILK-321 and 324-LMSTN / DLK-331 and (Zhou et al 2014 and internal data). The abundance of the AEP autocleavage peptides were quantified to measure a small, but statistically significant decrease in HCCF as compared to supernatant. This suggested that AEP has been converted to active form of protease after acid precipitation in HCCF sample, and protease activity would be elevated in this sample (FIG. 3C). FIG. 3D shows the sequence of CHO AEP legumain. Peptides detected by LC-MS are shown in bold.Proenzyme cleavage sites are indicated by blue stars on the highlighted tryptic peptides. CHO AEP residues N328, D308, and E314 correspond to N325, D305, and E311 in a universal AEP numbering proposed by Zhou et al 2014 for AEP from mouse, human and wild pig.Example 3. Addition of AEP inhibitor decreased the mAbl fragmentation

[0172] FIG. 4 shows the overall scheme in which the effect of an AEP inhibitor was evaluated. Addition of Fmoc-Ala-Glu-Asn-Lys-NH2 (AENK) inhibitor decreased the mABl light chain -NJ'L- fragmentation and LMW (-N^L- clipping abundance) percentage in an inhibitor concentration dependent manner (FIG. 5A).

[0173] FIGs. 6A-6B show the reduced reversed-phase LC-UV chromatograms of mAbl supernatant in pH 4.0 and 5.2 buffers with (+) and without (-) AEP inhibitor AENK. As shown in FIG. 6B, LMW light chain fragment (-N> L- clipping) abundance was monitored to track AEP induced cleavage. Addition of 0.3 mg / ml and 1.0 mg / rnL concentration of AENK inhibitor decreased the fragmentation of the antibody and LMW% in inhibitor concentration dependent manner.

[0174] FIGs. 7A-7B show the reduced reversed-phase LC-UV chromatograms of mAbl HCCF material in pH 4.0 and 5.2 buffers with (+) and without (-) AEP inhibitor AENK. As shown in FIG. 6B. LMW light chain fragment (-N> L- clipping) abundance was monitored to track AEP induced cleavage. Addition of 0.3 mg / ml and 1.0 mg / mL concentration of AENK inhibitor decreased the fragmentation of mAbl and LMW% in inhibitor concentration dependent manner.Example 4. Generation of AEP knockout cell lines.

[0175] To delete the endogenous asparaginyl endopeptidase (AEP), also known as legumain (LGMN). gene from the mAb l expressing production cell line using Cas-CLOVER gene editing technology, 5 single-guide RNAs targeting exon 1 on either the antisense or sense strand, respectively, were designed (Table 2). The guide RNA pairs were tested in individual transfections and showed variable cutting efficiencies. Guide RNA pair, rvsgmal and rvsgma3. showed low quality score. Pair rvsgmal and rvsgma5 showed no cutting. Pair rvsgma2 and rvsgma4 showed 64% indel with a knockout score of 48 (FIG. 8A). This sgRNA combination has a gap of 20 nucleotides. A pre-master cell bank (PMCB) vial of mAbl was thawed and transfected with Cas-CLOVER mRNA and the sgRNA pairs. Transfected pools were then single cell cloned 12 days after transfection to isolate clonal populations with the desired AEP deletion. After 14 days of growth, cherry picked clones were genotyped by Sanger sequencing the target AEP locus to identify AEP knockouts (KOs) for scale-up and banking. Out of the 144 wells exported, 88 (61%) clones showed evidence of growth and were cherry pi eked and adapted to suspension cultures. Robust growth was observed for 51 (56%) clones on day 14 post cherrypicking, and these clones were sent for genotyping via sanger sequencing. Genotyping results indicated a diverse array of mixed sequences; 27% of clones show no deletion, 65% of clones showed heterozygous deletions, and 8% of clones showed homozygous deletion. Based on genofyping and cell growth, 4homozy gous AEP KO clones were selected for further scale-up and characterization (FIG. 8B). Four clone sequence alignment with sequence of mAbl production cell line showed deletion of 13-29 base pair deletion between the two pair of sgRNA sequences. (FIG. 8C)Table 2. The guide RNA pairs used to target the region in exon 1 and their cutting efficiency.

[0176] Small-scale CTSD KO Clone Screening. Enzymes have diverse roles in cellular processes, and therefore, knockout of an enzyme may result in the disruption of an essential cellular function or may affect the production potential of a molecule expressing cell line. To test if the AEP KO clones do retain similar grow th and productivity characteristics as the original, wild-type production clone cell line, a comparative fed-batch evaluation was performed. 15-day fed batch evaluation of the clones along with positive control (parental mAbl production cell line AEP knockout pool) and a negative control (original wild-type production cell line for mAbl) demonstrated no impact on growth profile (VCD and Viability) of the cultures with a homozygous AEP gene knockout. (FIGs. 9A and 9B).Clones exhibited comparable titers as the mAbl production cell line (Table 3).Table 3. mAbl productivity in 15-day production run for mAbl production cell line (WT), mAbl knock out pool (KO Pool), and knock out clones. Comparable productivity for WT and knockout pool and knockout clones.

[0177] AEP KO resulted in a significant decrease in AEP (legumain) abundance in all AEP KO samples and down to zero (below the limit of detection) in clones. On the other hand, abundances of other CHO cell proteins remained similar in AEP KO and WT (FIGs. 11-12). This indicates that AEP deletion was performed with good precision and without impacting other genes / proteins. The proteomics and protein fragmentation data, together with the cell viability and titer data, indicate that the AEP KO pools and clones can be utilized for production of recombinant monoclonal antibodies.

[0178] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications, patents, and sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0179] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments.

Claims

What is claimed is:

1. A method for producing a recombinant protein, wherein said recombinant protein comprises an asparaginyl endopeptidase (AEP) cleavage site, comprising:(a) culturing a mammalian cell culture under a condition wherein said recombinant protein is expressed, and wherein said mammalian cell is an AEP knockout cell;(b) harvesting said recombinant protein from the culture; and(c) purifying said recombinant protein, wherein said purification comprises a step wherein the recombinant protein is subject to an acidic environment with a pH of 5 or less.

2. The method according to claim 1, wherein said purification process comprises an acidic precipitation step.

3. The method according to claim 1 or claim 2, wherein said harvesting in step (b) comprises harvesting the supernatant of the cell culture; and wherein said purification in step (c) comprises adding an acidic solution to the supernatant, such that the pH of the resulting supernatant is 5 or less.

4. The method according to claim 3, wherein the acidic solution comprises an acid selected from acetic acid, trichloroacetic acid, formic acid, phosphoric acid, sulfuric acid, citric acid, caprylic acid, and combinations of any of the foregoing.

5. The method according to any one of claims 1 to 4, wherein said purification in step (c) comprises subjecting the recombinant protein to an acidic environment for at least about 60 minutes.

6. The method according to any one of claims 1 to 5, wherein the recombinant protein is subject to an acidic environment with a pH of 4.6 or less.

7. The method according to any one of claims 1 to 6. wherein the recombinant protein is an immunoglobulin G (IgG).

8. The method according to any one of claims 1 to 7. wherein the recombinant protein is an IgGl, IgG2. IgG3, or IgG4.

9. The method according to any one of claims 1 to 8, wherein the recombinant protein is an IgGl or IgG2.

10. The method according to any one of claims 7 to 9. wherein said immunoglobulin comprises a P3-P2-P1-P1’-P2’-P3’ sequence motif, wherein Pl is Asn or Asp. Pl’ is any amino acid residue, and P2’ is a hydrophobic residue, and wherein the peptidyl bond between Pl and Pl ’ is cleaved by AEP.

11. The method according to any one of claims 1 to 10, wherein said immunoglobulin comprises a -NLA- sequence motif, and wherein the peptidyl bond between N and L is cleaved by AEP.

12. The method according to any one of claims 1 to 11, wherein the mammalian cell is a CHO cell or HEK cell.

13. The method according to any one of claims 1 to 12, wherein the mammalian cell is a CHO cell.

14. The method according to any one of claims 1 to 13, wherein both alleles encoding AEP of the mammalian cell have been knocked out.

15. The method according to any one of claims 1 to 14, wherein AEP is knocked out using CRISPR or using zinc-finger technology.

16. A mammalian cell comprising a genetic modification, wherein one or both alleles encoding asparaginyl endopeptidase (AEP) have been knocked out.

17. The mammalian cell of claim 16, wherein both alleles encoding AEP of the mammalian cell have been knocked out.

18. The mammalian cell of claim 16 or 17, wherein AEP has been knocked out using CRISPR or using zinc-fmger technology.

19. A method for producing a mammalian cell, wherein one or both alleles encoding asparaginyl endopeptidase (AEP) have been knocked out, comprising:(a) introducing to said cell two guide RNAs (gRNAs). first one comprising single guide RNA1 (sgRNAl), and second one comprising sgRNA2, wherein: (i) sgRNAl is complementary and binds to the antisense strand of AEP-encoding DNA and sgRNA2 is complementary and binds to the sense strand of AEP-encoding DNA; (ii) the binding site for sgRNAl and sgRNA2 are 14 to 40 nucleotides apart; (3) an NGGsequence immediately follows the 3'-end of sgRNAl; and (4) an NGG sequence immediately follows the 3 ’-end of sgRNA2; and(b) introducing to said cell a nucleic acid encoding a deactivated Cas9 (dCas9) and aClo51.

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