RECOMBINANTLY ENGINEERED, LIPASE/ESTERASE-DEFICIENT MAMMALIAN CELL LINES

Abstract

Mammalian cell lines with reduced expression and/or activity of lipases/esterases, and methods of producing the same are provided. Also provided are compositions comprising polysorbate and recombinant proteins produced in said mammalian cells which have improved polysorbate stability.

Claims

1. A recombinantly engineered mammalian cell having reduced expression and/or reduced activity of at least one endogenous host cell protein (HCP) palmitoyl-protein thioesterase (PPT) and at least one other endogenous HCP selected from the group consisting of a lipoprotein lipase, a lysosomal acid lipase, a phospholipase D, and a phospholipase A2 comprising a disrupted or inactivated gene encoding the palmitoyl-protein thioesterase and a disrupted or inactivated gene encoding at least one HCP selected from the group consisting of a lysosomal acid lipase protein, a lipoprotein lipase protein, a phospholipase D, and a phospholipase A2 protein.

2. The cell of claim 1 wherein the palmitoyl-protein thioesterase is PPT1.

3. The cell of claim 2 wherein at least one inactivated gene encoding a HCP is selected from the group consisting of LAL, LPL, PLD3 and LPLA2.

4. The cell of claim 3, wherein the cell comprises a modification in a coding sequence of a polynucleotide encoding the LAL protein, the LPL protein, the LPLA.sub.2 protein, and the PPT1 protein.

5. The cell of claim 4, wherein the cell comprises a modification in a coding sequence of a polynucleotide encoding the LAL protein, the LPL protein, the LPLA.sub.2 protein, and the PPT1 protein, wherein the modification decreases the expression level of the LAL protein, the LPL protein, the LPLA.sub.2 protein, and the PPT1 protein in a cell having the modification relative to the expression level of a cell without any of said modifications.

6. The cell of claim 5, wherein the cell does not express detectable levels of the LAL protein, the LPL protein, the LPLA.sub.2 protein, and the PPT1 protein.

7. The cell of claim 6, wherein the modification comprises a nucleotide insertion or deletion within exon 1 or 2 of the coding sequence of the polynucleotide encoding the particular protein.

8. The cell of claim 7, wherein the modification comprises: a) a nucleotide insertion or deletion within exon 1 of the coding sequences of the polynucleotide encoding the LPL, the LPLA.sub.2, and PPT1 proteins, and b) a nucleotide insertion or deletion within exon 2 of the coding sequence of the polynucleotide encoding the LAL protein.

9. The cell of claim 8, wherein the PPT1 protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:1.

10. The cell of claim 9, wherein the modification comprises a nucleotide insertion or deletion within SEQ ID NO:8.

11. The cell of claim 8, wherein the LAL protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:2.

12. The cell of claim 11, wherein the modification comprises a nucleotide insertion or deletion within SEQ ID NO:7.

13. The cell of claim 8, wherein the LPL protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:3.

14. The cell of claim 13, wherein the modification comprises a nucleotide insertion or deletion within SEQ ID NO:6.

15. The cell of claim 8, wherein the LPLA.sub.2 protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:4.

16. The cell of claim 15, wherein the modification comprises a nucleotide insertion or deletion within SEQ ID NO:5.

17. The cell of claim 16, wherein the modification comprises a nucleotide insertion or deletion within exon 2, exon 3, or exon 4 of the coding sequence of the polynucleotide encoding a protein from the list comprised of: PPT1, LAL, LPL, and LPLA.sub.2.

18. The cell of claim 17 further comprising a polynucleotide encoding one or more bioproducts.

19. The cell of claim 18, wherein the bioproduct is selected from the group consisting of an antibody, an antibody heavy chain, an antibody light chain, an antigen-binding fragment, an antigen-binding protein, protein-protein fusion and an Fc-fusion protein.

20. The cell of claim 19, wherein the cell produces a protein A-binding fraction having substantially reduced polysorbate degradation activity relative to the polysorbate degradation activity of a cell without any of the modifications.

21. The cell of claim 20, wherein the reduction in degradation of intact polysorbate is greater than 30%.

22. The cell of claim 20, wherein the reduction in degradation of intact polysorbate is greater than 40%.

23. The cell of claim 21, wherein the cell is a CHO cell.

24. The cell of claim 23, wherein the cell is a CHO-K1 cell, a CHOK1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knock-out cell (glutamine synthetase), a CHOK1SV FUT8 knock-out cell, a CHOZN, or a CHO-derived cell.

25. A method of producing a bioproduct comprising the steps of: (a) obtaining a sample comprising a bioproduct and a plurality of host cell proteins from a host cell modified to produce reduced levels of PPT1 compared to an unmodified cell; and (b) subjecting the sample to at least one purification step to remove at least one host cell protein.

26. The method of claim 25, wherein the plurality of host cell proteins (a) does not comprise a detectable amount of a PPT1 protein; and (b) does not comprise a detectable amount of at least one other lipase or esterase.

27. The method of claim 26, wherein the host cell comprises: a) a modification in a coding sequence of a polynucleotide encoding a PPT1 protein; and b) a modification in a coding sequence of a polynucleotide encoding a fatty acid hydrolase selected from the group consisting of LAL, LPL, LPLA2, PLD3, or a combination thereof.

28. The method of claim 27, wherein the purification step is protein A affinity (PA) chromatography or another affinity chromatography method, cation exchange (CEX) chromatography, anion exchange (AEX) chromatography or hydrophobic interaction chromatography (HIC).

29. A process for reducing polysorbate degradation in a protein formulation comprising the steps of: (a) modifying a host cell to reduce or eliminate the expression of PPT1 protein; (b) modifying the host cell to reduce or eliminate the expression of LAL, LPL, PLD3, and/or LPLA.sub.2; (c) transfecting the cell with a polynucleotide encoding a bioproduct; (d) extracting a protein fraction comprising the protein of interest from the host cell; (e) contacting the protein fraction with a chromatography media which is PA chromatography or another affinity chromatography method, CEX chromatography, AEX chromatography or HIC; and (f) collecting the protein of interest from the media; (g) combining the bioproduct with a fatty acid ester; and (h) optionally, adding a buffer; and (i) optionally, adding one or more pharmaceutically acceptable carriers, diluents, or excipients.

30. A process for reducing aggregation or particle formation in a protein formulation comprising the steps of: (a) modifying a host cell to reduce or eliminate the expression of PPT1 protein; (b) modifying the host cell to reduce or eliminate the expression of LAL, LPL, PLD3, and/or LPLA.sub.2; (c) transfecting the cell with a polynucleotide encoding a bioproduct of interest; (d) extracting a protein fraction comprising the protein of interest from the host cell; (e) contacting the protein fraction with a chromatography media which is PA chromatography or another affinity chromatography method, CEX chromatography, AEX chromatography or HIC; and (f) collecting the protein of interest from the media; and (g) combining the protein of interest with a fatty acid ester; and (h) optionally, adding a buffer; and (i) optionally, adding one or more pharmaceutically acceptable carriers, diluents, or excipients.

31. A process for producing a stable formulated bioproduct comprising: (a) modifying a host cell to reduce or eliminate the expression of PPT1 protein; (b) modifying the host cell to reduce or eliminate the expression of LAL, LPL, PLD3, and/or LPLA.sub.2; (c) transfecting the cell with a polynucleotide encoding a bioproduct; (d) extracting a protein fraction comprising the bioproduct from the host cell; (e) contacting the protein fraction with a chromatography media which is PA chromatography or another affinity chromatography method, CEX chromatography, AEX chromatography or HIC; (f) collecting the bioproduct from the media; (g) combining the bioproduct with a fatty acid ester; (h) optionally, adding a buffer; and (i) optionally, adding one or more pharmaceutically acceptable carriers, diluents, or excipients.

32. The process of claim 29, wherein the step of modifying the host cell to reduce or eliminate the expression of PPT1 comprises inserting or deleting at least one nucleotide within exon 2, exon 3 or exon 4 of a polynucleotide encoding the PPT1 protein.

33. The process of claim 32, wherein the polynucleotide encoding the PPT1 protein comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO:1.

34. The process of claim 33, wherein the expression and/or activity of any of the phospholipases produced by the cell is reduced.

35. The process of claim 34, wherein the reduced expression and/or activity is determined by assaying for lipolytic activity.

36. A pharmaceutical composition comprising a polysorbate and a bioproduct produced by the mammalian cell of claim 32.

37. A pharmaceutical composition comprising a polysorbate and a bioproduct produced by the process of claim 35.

38. The pharmaceutical composition of claim 37 wherein the bioproduct is selected from the group consisting of tanezumab, lebrikizumab, mirikizumab, solanezumab, donanemab, zagotenemab, ramucirumab, galcanezumab, ixekizumab, dulaglutide, necitumumab, olaratumab, cetuximab, an angiopoietin 2 mAb, an insulin-Fc fusion protein, CD200R agonist antibody, epiregulin/TGFα mAb, ANGPTL 3/8 antibody, a BTLA antibody agonist, a CXCR1/2 ligands antibody, a GDF15 agonist, an IL-33 antibody, a PACAP38 antibody, a PD-1 agonist antibody, pGlu-Abeta, also called N3pG Abeta mAb, a TNFα/IL-23 bispecific antibody, an anti-alpha-synuclein antibody, CD226 agonist antibody, MCT1 antibody, a SARS-CoV-2 neutralizing antibody, an FcgRIIB antibody, an IL-34 antibody, a CD19 antibody, a TREM2 antibody, and a relaxin analog; and polysorbate wherein the bioproduct was produced by the recombinant mammalian cells of the present invention.

39. A bioproduct made by the process of claim 35.

Description

BRIEF DESCRIPTION OF FIGURES

[0119] FIG. 1: A graph depicting the temperature-dependent degradation of PS80 mono-oleate ester in the presence of PPT1 over time, which demonstrates that PPT1 degrades PS80 over time in a temperature-dependent manner.

[0120] FIG. 2: A graph depicting the degradation of PS80 mono-oleate ester over time in formulated mAb samples: control (A), and spiked separately with (B) LAL—1 ppm, (C) LPL—1 ppm, (D) PPT1—1 ppm, and (E) LPLA2—0.1 ppm, demonstrating that PS80 mono-oleate ester present in the formulation degrades over time to a greater extent in the presence of these proteins than the formulated mAb control.

[0121] FIG. 3: A graph depicting the degradation of PS80 mono-oleate ester over time in control sample (A) and in the presence of 0.25 UN/mL PLD4 (B), 2.5 UN/mL PLD4 (D), 0.25 UN/mL PLD7 (C), and 2.5 UN/mL PLD7 (E). This data qualitatively demonstrates the capacity for PLD family members to degrade PS80 over time.

[0122] Without limiting the scope of the invention, the following preparations and examples are provided for those of ordinary skill in the art a means of making and using the methods and compositions described herein.

EXAMPLES

Example 1

Characterization of Polysorbate Hydrolytic Activity of PPT1

Polysorbate Degradation Analysis by Liquid Chromatography-Mass Spectrometry (LCMS)—General Procedure A

[0123] LCMS analyses are performed on a Waters ACQUITY UPLC (I class) equipped with a Waters SYNAPT® G2-Si mass spectrometer; column: Agilent PLRP-S 2.1×50 mm, 1000 Å, 5 μm particle size; mobile phase: A—0.05% trifluoroacetic acid (TFA) in water, B—0.04% TFA in acetonitrile. Standard solutions are prepared with 2% PS80 and 10 mM citrate buffer to get 0.001, 0.002, 0.005, 0.01, 0.025, 0.05% PS80 solutions. Standard curves of prepared PS80 solutions in 10 mM citrate buffer are obtained in order to quantify intact PS80 in samples by LCMS extracted ion chromatograms for polysorbate mono-oleate. Using the standard curves, the relative percent (%) of intact PS80 as a mono-oleate ester for each sample is calculated against time=zero.

Example 1a

Polysorbate 80 Degradation in the Presence of PPT1

[0124] Samples of polysorbate 80 (PS80) and PPT1 are prepared as follows: 0.5 mL of 0.02% w/v PS80 in 10 mM citrate buffer (pH 6) is mixed with 5.6 μL of a 0.3 mg/mL solution of PPT1 (prepared by recombinant expression) and samples are kept at 4, 15, 25, and 35° C. for the duration of the study. Samples (50 μL) of these solutions are taken at time intervals and mixed with 5 μL of 5% formic acid in water for LCMS analysis. The percent of remaining intact PS80 as a mono-oleate ester is monitored by LCMS over time using General Procedure A. These data are shown in FIG. 1 and demonstrate that PPT1 degrades PS80 over time in a temperature-dependent manner.

Example 1b

Polysorbate 80 Degradation in mAb Formulation Samples Spiked with LAL, LPL, PPT1, and LPLA2

[0125] Samples of a formulated mAb (Antibody 1, 100 mg/mL in 20 mM sodium acetate buffer, pH 5.0, with 0.03% w/v PS80) are spiked separately with 1 ppm LAL, LPL, and PPT1, and 0.1 ppm LPLA2 (obtained from recombinant expression). The samples are incubated at 37° C. for the duration of the study. Each sample is diluted with 20 mM sodium acetate buffer in 1:2 ratio and then analyzed by LCMS using General Procedure A. The percent of remaining intact PS80 as a mono-oleate ester over time is shown in Table 1 and FIG. 2.

TABLE-US-00001 TABLE 1 Relative Percent (%) vs the Time Zero of Intact PS80 in Samples of Antibody 1 Spiked with LAL, LPL, PPT1, and LPLA2 PS80 mono-oleate ester remaining (average of relative percent (%) ± standard deviation) after: 0 days 2 days 5 days 7 days 14 days Antibody 1 control 100 90.7 ± 2.3 88.7 ± 2.9 85.1 ± 2.0 84.2 ± 1.3 Antibody 1 spiked 100 64.6 ± 2.2 52.9 ± 2.2 53.9 ± 2.9 34.1 ± 0.1 with LAL, 1 ppm Antibody 1 spiked 100 13.9 ± 1.0  7.6 ± 0.4  7.5 ± 0.5  5.2 ± 0.3 with LPL, 1 ppm Antibody 1 spiked 100 69.5 ± 0.7 41.6 ± 6.2 43.5 ± 1.6 17.1 ± 2.8 with PPT1, 1 ppm Antibody 1 spiked 100 21.1 ± 0.6 16.0 ± 0.9 15.3 ± 7.7  0.2 ± 0.0 with LPLA2, 0.1 ppm Note: All results in Table 1 represent n = 2
These data demonstrate that PS80 mono-oleate ester present in the formulation degrades over time to a greater extent in the presence of these proteins than the formulated mAb control.

[0126] Together the data in this example demonstrate the ability of these proteins (LAL, LPL, PPT1, and LPLA2) to degrade PS80 in solution over time.

Example 2

Identification of PPT1 in an Fc-Fusion Protein Formulation

[0127] Two separate culture batches of an Fc-fusion protein (Fc-Fusion Protein 1) are subjected to Protein A chromatography. Aliquots (25 μL) of the Protein A mainstreams are mixed with of 1M Tris-HCl buffer, pH 8 (5 μL), Barnstead water (172 μL), a protein standard mixture (0.8 μL), and 2.5 mg/mL bovine r-trypsin (2 μL). The samples are incubated at 37° C. for 16 hours. The samples are mixed with 2 μL of a 50 mg/mL dithiothreitol (DTT) solution and then heated at 90° C. for 10 min. The samples are centrifuged at 10,000 g for 2 minutes and the supernatants are transferred into vials. The samples are then acidified with 5% TFA in H.sub.2O (5 μL) and analyzed by LCMS. LCMS analysis is performed on a Waters ACQUITY UPLC equipped with a ThermoFisher Q Exactive™ Plus mass spectrometer; column: Waters UPLC CSH C18, 2.1×50 mm, 1.7 μm particle size; mobile phase: A—0.10% formic acid (FA) in water, B—0.10% FA in acetonitrile, with the column submerged in ice water. In this experiment PPT1 is identified in the samples of Fc-Fusion Protein 1 post-Protein A purification by a non-target proteomics (DDA) approach at 0.5±0.1 ppm (n=2).

Example 3

Generation of a Recombinantly Engineered LPLA2, LAL, LPL, and PPT1 Knockout CHO Cell Line

[0128] Unless otherwise noted, the cell culture media used refers to serum-free cell culture media supplemented with 8 mM glutamine. Additionally, unless otherwise noted, the mammalian cells used are a glutamine synthase deficient CHO (GS-CHO) cell line.

[0129] Engineering of cell lines is accomplished through the use of custom-made zinc-finger nuclease (ZFN) reagents designed to be specific for each target HCP gene, constructed by Sigma Aldrich (CompoZr® Custom Zinc Finger Nuclease, Cat. No. CSTZFN, Sigma Aldrich, St. Louis, Mo.). The ZFN binding/cutting region nucleic acid sequences for LPLA2, LPL, LAL, and PPT1 are given in Table 2.

TABLE-US-00002 TABLE 2 ZFN binding/cutting regions for LPLA2, LPL, LAL, and PPT1. SEQ Gene ZFN bind/cut nucleic Bind/ ID target acid sequence (cut sequence cut NO: HCP: lower-case and italicized): exon: 5 LPLA2 TGGATCGCCATCACCTCActtgtcGCG 1 CGACCCAGCTCCGGAG 6 LPL AGCAAAGCCCTGCTCCTGGtggctCTG 1 GGAGTGTGGCTCCAG 7 LAL TACTGGGGATACCCGAGTgaggaGCAT 2 ATGATCCAGAC 8 PPT1 CGCCTTCGCTGACACCGCtggtgATCT 1 GGcATGGGATGGGTA

Preparation of Cells for Gene Disruption—General Procedure B

[0130] Vials containing cells are thawed in a 36° C. water bath until only a sliver of ice remains. The cells are seeded into cell culture media in shake-flask culture. The culture of the parental cell line is sub-cultured into cell culture media and is maintained and passaged on a 3-day/4-day schedule. Cell cultures are seeded at a 0.2×10.sup.6 vc/mL seed density in 30 mL appropriate maintenance medium, as noted above. On the day of transfection, the cells are counted and an appropriate volume of cells is harvested.

ZFN Transfection and Bulk Culture Recovery—General Procedure C

[0131] ZFN transfections are performed using the Nucleofector™ technology and associated cGMP Nucleofector™ Kit V (Cat. No. VGA-1003, Lonza, Basel, Switzerland). Briefly described, enough cells for single Nucleofection reactions (2-4.5×10.sup.6 vc) are collected by centrifugation. Following complete removal of the supernatant, the cell pellet is suspended in 100 μL of Nucleofector™ solution V, with supplement added, according the manufacturer's protocol. The suspended cells are gently mixed by trituration and transferred to a vial containing an aliquot of the ZFN mRNA [part of the custom ZFN kit generated by Sigma Aldrich (St. Louis, Mo.)]. The cell/mRNA mixture is then transferred to a 2 mm cuvette provided in the Nucleofector™ kit, the cuvette is inserted into the Nucleofector™ device, and the cells are electroporated. Following electroporation, the cells rest at room temperature in the cuvette for 30-60 seconds, and then they are transferred using a sterile transfer pipet to a well in a labeled 6-well plate (Falcon Cat. no. 351146, Corning, Durham, N.C.) containing 3 mL cell culture media. The transfected cells are maintained in the 6-well plate, static, in a humidified incubator for 1-4 days at 36° C., 6% CO.sub.2, after which they are transferred to shake-flask culture in cell culture media, 36° C., 6% CO.sub.2, shaking 125 rpm, until the viability is >90%. Once the cells are recovered completely from transfection (as measured by viability in shake-flask culture), the bulk culture is single-cell sorted using FACS technology.

[0132] The ZFN transfections for each target HCP may be performed a single time prior to single-cell sorting. Alternatively, the ZFN transfections for any particular target HCP may performed two times, with complete cell recovery prior to the second ZFN transfection. More than one round of ZFN transfection may increase the number of cells containing a bi-allelic mutation in the respective target HCP, making screening more efficient.

Detection of ZFN-Mediated Target HCP Sequence Modifications in Bulk Cultures—General Procedure D

[0133] Two to seven days post-transfection, cells from the partially-to-fully recovered ZFN bulk cultures are harvested for evaluation to assess the activity of the transfected ZFN. The Surveyor® Mutation Detection Assay (MDA) (Transgenomic Inc., Omaha, Neb.) is used to detect the efficiency of the ZFN procedure in generating modifications at the target HCP site, according to the manufacturer's protocol. Briefly, the ZFN-binding region is PCR amplified using primers provided in the CompoZr® Custom Zinc Finger Nuclease kit (Sigma, St. Louis, Mo.). The PCR products are then denatured and re-annealed. The Cel-I endonuclease (Surveyor Nuclease S) provided in the MDA kit is used to detect DNA mismatch “bubbles”, derived from the annealing of PCR products consisting of the native or wild-type sequence and those that contain indels, as Cel-I will recognize these “bubbles” of mismatch and cleave the DNA. After the Cel-I digest, products are then resolved on a 2% or 4% TBE agarose gel (Reliant Gel, Lonza, Basel, Switzerland). In the absence of DNA mismatch “bubbles”, no DNA cleavage will occur and only one band will be present, representing the PCR product. If any non-homologous end-joining (NHEJ) occurred, representing ZFN activity, cleavage products will be observed on the gel in the form of two (or more) bands. Only those ZFN bulk cultures that show a positive response in the MDA are forward-processed to single-cell sorting.

Single-Cell Sorting by Fluorescence-Activated Cell Sorting—General Procedure E

[0134] The recovered bulk culture is sorted via Fluorescence-Activated Cell Sorting (FACS) technology. The protocols and methods for the single-cell cloning are well-known in the art. For cloning, a cell sorter (MoFlo™ XDP, Beckman Coulter) is used to identify and sort single, viable cells by measuring laser diffraction in the forward and side-scatter directions, according to methods which are well-known in the art (see, for example, Krebs, L., et al. (2015) “Statistical verification that one round of fluorescence-activated cell sorting (FACS) can effectively generate a clonally-derived cell line.” BioProcess J 13(4): 6-19).

[0135] Cells are sorted into 96-well microtiter plates (Falcon, catalog number 35-3075) containing animal-component free sort medium (Ex-Cell CHO cloning media, SAFC C6366)+20% conditioned cell culture medium+phenol red (Sigma P0290)). To prepare conditioned cell culture medium, parental cells are seeded at a density of 1×10.sup.6 vc/mL into a cell culture medium without glutamine and incubated in a shake-flask at 36° C., 6% CO.sub.2, 125 rpm for 20-24 h. The culture is centrifuged to remove cells and the conditioned media is filtered through a sterile 0.22 μm filter. Seven to ten days post single-cell sort, all the plates are fed with 50 μL cell culture media per well. On day 14-15 post single-cell sort, the plates are analyzed for clonal outgrowth. Outgrowth is determined by imaging of the sort plates using a CloneSelect Imager (Molecular Devices, Sunnyvale, Calif.) or manually with the aid of a mirror and/or by observation of a medium color change from red to orange/yellow.

Screening Clonally-Derived Cell Lines for ZFN-Mediated Target HCP Sequence Modifications—General Procedure F

[0136] Clonally-derived cell lines (CDCLs) are picked from 96-well plates that originate from the recovered ZFN bulk culture as they become a visible colony and are transferred to deep 96-well plates (Greiner, Catalog No. 780271) containing cell culture medium. Clonally-derived cell lines are consolidated into deep-well plates containing 150 μL cell culture medium. The cultures are maintained in cell culture medium under static conditions on a 3-day/4-day feed/pass schedule until screening and characterization is complete.

[0137] Clonally-derived cell lines (CDCLs) are screened for indels using the Surveyor® MDA. Genomic DNA is isolated from each cell line using the Promega Wizard® SV 96 Genomic DNA Purification Kit (cat. no. A2371, Promega, Madison, Wis.), according to the manufacturer's protocol. The ZFN PCR reactions are performed using the Phusion® High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, Mass.), according to manufacturer's protocol. MDA digestion products are resolved on 2% TBE agarose gels. Cell lines which have been identified that are positive in the MDA are characterized through either General Procedure G or General Procedure H.

Characterizing Indels in CDCLs using RT-PCR—General Procedure G

[0138] CDCLs are characterized by sequencing of the ZFN PCR products using a target gene RT-PCR reaction. Total RNA is isolated from each potential KO cell line using the RNeasy Micro Kit (Qiagen, Cat. No. 74004, Germantown, Md.), according to manufacturer's protocol. Reverse transcription reactions are done using the SuperScript™ III First-Strand Synthesis System for RT-PCR (cat. no. 18080-051, Invitogen, Carlsbad, Calif.), according to manufacturer's protocol, followed by PCR reactions using the Phusion® High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, Mass.), according to manufacturer's protocol. The RT-PCR products are resolved on 1% TAE agarose gels, identifying cell lines with altered RT-PCR products. The cell line chosen for forward-processing lacks a RT-PCR product and does not contain the target HCP protein by LCMS.

Characterizing Indels in CDCLs Using Next-Generation Sequencing (NGS)—General Procedure H

[0139] MDA-positive CDCLs are consolidated into 96-well deep-well plates for further maintenance. When consolidating, those cell lines that show “off-normal” PCR and/or MDA results are characterized using next-generation sequencing (NGS) provided by GENEWIZ. Cell lines containing acceptable bi-allelic indels in the target HCP gene locus are evaluated by LCMS, carrying forward a cell line which does not contain the target HCP protein.

Scaling and Banking Knockout Cell Lines—General Procedure I

[0140] Those CDCLs that, based on the initial screen/characterization work, warrant further evaluation are scaled from the 96-well deep-well plates (DWPs) to shake-flasks, and research cell banks (RCB) are generated. From the DWP, cells from the appropriate wells are transferred to an appropriately labeled well in a 6-well plate containing 3 mL cell culture medium. The scaling CDCLs are maintained in the 6-well plate, static, in a humidified incubator for 3 to 4 days at 36° C., 6% CO.sub.2, after which they are transferred to shake-flasks, containing 15 mL of cell culture medium, 36° C., 6% CO.sub.2, shaking at 125 rpm. The shake-flask cultures are passed at least one time to build suitable cell mass for banking. For each cell line, a 3-10 vial RCB is generated with 10-13×10.sup.6 vc per vial in Freezing Menstrum (90:10 cell culture medium:DMSO). The vials are placed in a styrofoam rack “sandwich” at −80° C. for at least 24 h to allow for a controlled-rate freezing of the cells. Once the vials are completely frozen they are stored at −80° C.

Example 3a

LPLA2 Knockout CHO Cell Line

[0141] CHO cells are prepared for gene disruption according to General Procedure B. The cells are then subjected to a single ZFN transfection and bulk culture recovery according to General Procedure C. Using General Procedure D, sequence modifications in bulk culture are detected. Bulk cultures showing a positive response in the MDA are forward-processed to single-cell sorting according to General Procedure E. Clonally-derived cell lines obtained therefrom are screened for target HCP sequence modifications according to General Procedure F. Indels are characterized according General Procedure G, and a cell line is chosen which does not contain detectable amounts of the LPLA2 protein by LCMS. An RCB is generated according to General Procedure I to give an LPLA2 knockout CHO cell line.

Example 3b

LPLA2/LPL Knockout CHO Cell Line

[0142] LPLA2 knockout CHO cells from Example 3a are prepared for gene disruption according to General Procedure B. The cells are then subjected to two ZFN transfections and bulk culture recovery according to General Procedure C. Using General Procedure D, sequence modifications in bulk culture are detected. Bulk cultures showing a positive response in the MDA are forward-processed to single-cell sorting according to General Procedure E. Clonally-derived cell lines obtained therefrom are screened for target HCP sequence modifications according to General Procedure F. Indels are characterized according General Procedure H and a cell line is chosen which does not contain detectable amounts of the LPL protein by LCMS. An RCB is generated according to General Procedure Ito give an LPLA2/LPL knockout CHO cell line.

Example 3c

LPLA2/LPL/LAL Knockout CHO Cell Line

[0143] LPLA2/LPL knockout CHO cells from Example 3b are prepared for gene disruption according to General Procedure B. The cells are then subjected to two ZFN transfections and bulk culture recovery according to General Procedure C. Using General Procedure D, sequence modifications in bulk culture are detected. Bulk cultures showing a positive response in the MDA are forward-processed to single-cell sorting according to General Procedure E. Clonally-derived cell lines obtained therefrom are screened for target HCP sequence modifications according to General Procedure F. Indels are characterized according to General Procedure H and a cell line is chosen which does not contain detectable amounts of the LAL protein by LCMS. An RCB is generated according to General Procedure Ito give an LPLA2/LPL/LAL knockout CHO cell line.

Example 3d

LPLA2/LPL/LAL/PPT1 Knockout CHO Cell Line

[0144] LPLA2/LPL/LAL knockout CHO cells from Example 3c are prepared for gene disruption according to General Procedure B. The cells are then subjected to two ZFN transfections and bulk culture recovery according to General Procedure C. Using General Procedure D, sequence modifications in bulk culture are detected. Bulk cultures showing a positive response in the MDA are forward-processed to single-cell sorting according to General Procedure E. Clonally-derived cell lines obtained therefrom are screened for target HCP sequence modifications according to General Procedure F. Indels are characterized according General Procedure H, however none of the cell lines contain bi-allelic mutations in the targeted PPT1 region. Cell lines containing mono- or bi-allelic indels are evaluated by LCMS, carrying forward a cell line which does not contain detectable amounts of the PPT1 protein by LCMS evaluation. An RCB is generated according to General Procedure Ito give an LPLA2/LPL/LAL/PPT1 knockout CHO cell line.

Example 4

Comparison of Polysorbate Stability in Formulated mAbs Expressed in a LPLA2/LPL/LAL/PPT1 Knockout CHO Cell Line vs. Control

[0145] An Fc-fusion protein (Fc-Fusion Protein 1) and an antibody (Antibody 2) are produced from product expressing CHO cell lines with LPLA2, LPL, LAL, and PPT1 knocked out (referred to as “lipase/esterase KO cell line”) and also product expressing CHO cell lines without LPLA2, LPL, LAL, or PPT1 knockouts as a control. Fc-Fusion Protein 1 is processed through Protein A chromatography, low pH viral inactivation, anion exchange chromatography (AEX), cation exchange (CEX) chromatography, and tangential flow filtration (TFF) concentration prior to formulation with 0.02% PS80. Antibody 2 is processed through Protein A chromatography, low pH viral inactivation, CEX chromatography, and TFF concentration prior to formulation with 0.02% PS80. Formulated samples of Fc-Fusion Protein 1 and Antibody 2 are kept at 25° C. for the duration of the study and used directly for LCMS analysis, using General Procedure A to monitor the percent of remaining intact PS80 as a mono-oleate ester over time. The results are listed in Table 3 and indicate that PS80 in Fc-Fusion Protein 1 and Antibody 2 produced using the KO cell line are more stable than the control samples.

TABLE-US-00003 TABLE 3 Relative Percent (%) vs the Time Zero of Intact PS80 in Samples of Antibody 2 and Fc-Fusion Protein 1 PS80 mono-oleate ester remaining (average of relative percent (%) ± standard deviation) after: 0 weeks at 2 weeks at 4 weeks at 8 weeks at Sample: 25° C. 25° C. 25° C. 25° C. Antibody 2 from 100 83 ± 3 77 ± 2 69 ± 1 lipase/esterase KO cell line Antibody 2 control 100 35 ± 8 24 ± 5 15 ± 4 Fc-Fusion Protein 1 100 104 ± 5  113 ± 6  93 ± 4 from lipase/esterase KO cell line Fc-Fusion Protein 1 100 68 ± 4 54 ± 4 33 ± 7 control Note: All results represent n = 3

Example 5

Identification of PLD3 in a Monoclonal Antibody Formulation

[0146] Samples containing 1 mg of Antibody 3 which have been processed through Protein A capture, low pH viral inactivation, anion exchange (AEX) chromatography, and concentration by tangential flow filtration (TFF) to a concentration of 150 mg/mL are mixed with Tris-HCl buffer (1 M, pH 8, 5 μL) and water to achieve a volume of 195 μL. Each solution is treated with 5 μL of tryspin and protein standard mixture (20 μL of 2.5 mg/mL r-bovine trypsin, 20 μL of a protein standard mixture and 60 μL of water) at 37° C. overnight. Each sample is mixed with 1,4-dithiothreitol (DTT, 50 mg/mL, 2 μL) and heated to 90° C. for 10 min, observing a white precipitate. The samples are then centrifuged at 13000g for 2 min and the supernatant is transferred into a HPLC vial. The samples are acidified with 5 μL of 10% formic acid in water before LCMS analysis essentially as described for Example 2. In this experiment PLD3 is identified in the samples of Antibody 3 at 17±6 ng/mg (n=2) of Antibody 3.

Example 6

Characterization of Polysorbate Hydrolytic Activity of PLD4 and PLD7

[0147] PLD4 and PLD7, like PLD3, are phospholipase D family members. The hydrolytic activity of PLD4 and PLD7 is assessed in a manner that is essentially as described in Example 1. Samples containing 0.02% PS80 are incubated with 0.25 and 2.5 units per milliliter (UN/mL) of PLD4 and PLD7 at 35° C., and the percent of remaining intact PS80 as a mono-oleate ester is monitored by LCMS over time using General Procedure A. After 35 h incubation under these conditions, PS80 is >30% and >80% hydrolyzed in the presence of 2.5 UN/mL PLD4 and PLD7, respectively. These data are shown in FIG. 3, and qualitatively demonstrate the capacity for PLD family members to degrade PS80 over time.