PRODUCTION CELLS

Abstract

Herein are reported modified mammalian cells wherein the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes has been reduced or eliminated as well as methods for the recombinant production of a heterologous polypeptide using a modified mammalian cell according to the current invention. Further reported are use of the reduction of the expression of the genes for increasing volumetric productivity, increasing cell volume, increasing viability and increasing the possible cultivation time without cell split.

Claims

1. A modified mammalian cell wherein the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes has been reduced or eliminated.

2. The modified mammalian cell according to claim 1, wherein the expression of the MYC, SIRT-1 and ICAM-1 genes, or the MYC, BAX, BAK and SIRT-1 genes, or the MYC, BAX, BAK, ICAM-1 and SIRT-1 genes has been reduced or eliminated.

3. The modified mammalian cell according claim 1, wherein the reduction or elimination is by gene knockout.

4. The modified mammalian cell according to claim 1, wherein the modified mammalian cell comprises one or two or more targeted integration landing sites.

5. The modified mammalian cell according to claim 1, wherein the modified mammalian cell comprises one or two or more targeted integration landing sites, whereby in one targeted integration landing site one or more nucleic acids have been integrated that encode a mono- or multispecific antibody.

6. The modified mammalian cell according to claim 1, wherein the reduction or elimination of the expression of the MAY, BAK, BAX, ICAM-1 or/and SIRT-1 genes has been effected after the stable introduction of one or more nucleic acids encoding a heterologous protein.

7. A modified mammalian cell according to claim 1, wherein the modified cell is a modified CHO or CHO-K1 cell.

8. A modified mammalian cell according to claim 1, wherein the modified cell is a modified HEK 293, HEK293T, BHK, A549 or HeLa cell.

9. A method for the recombinant production of a protein comprising the steps of a) cultivating a modified mammalian cell according to claim 1 comprising one or more nucleic acids encoding the protein in a cultivation medium under conditions suitable for the expression of the protein, b) recovering the protein from the modified mammalian cell or the cultivation medium, c) optionally purifying the protein with one or more chromatography steps, and thereby recombinantly producing the protein.

10. The method according to claim 9, wherein the cultivating is for 6 to 16 days.

11. The method according to claim 9, wherein the cultivating is started with a cell density of 5*10E6 cells/ml or more.

12. The method according to claim 9, wherein the cultivating is a fed-batch cultivating with feeding at least on days 1, 4, 7 and 10.

13. The method according to claim 9, wherein the modified mammalian cell is a modified CHO or CHO-K1 cell.

14. The method according to claim 9, wherein the modified mammalian cell is a modified HEK 293, HEK293T, BHK, A549, or HeLa cell

15. Use of the reduction or elimination of the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes in a modified mammalian cell expressing a recombinant polypeptide for increasing the volumetric productivity of the modified mammalian cell.

16. Use of the reduction or elimination of the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes in a modified mammalian cell for increasing cell volume.

17. Use of the reduction or elimination of the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes in a modified mammalian cell expressing a recombinant polypeptide for increasing bioprocess viability.

18. Use of the reduction or elimination of the expression of the MYC gene and one or more of the BAK, BAX, SIRT-1 and ICAM-1 genes in a modified mammalian cell expressing a recombinant polypeptide for increasing/extending the cultivation time without cell-split of the modified mammalian cell.

19. The use according to claim 15, wherein the expression of the MYC, SIRT-1 and ICAM-1 genes, or the MYC, BAX, BAK and SIRT-1 genes, or the MYC, BAX, BAK, ICAM-1 and SIRT-1 genes has been permanently reduced or eliminated.

20. The use according to claim 15, wherein the modified mammalian cell is a modified CHO or CHO-K1 cell.

21. The method according to claim 15, wherein the modified mammalian cell is a modified HEK293, HEK293T, BHK, A549, or HeLa cell.

Description

DESCRIPTION OF THE FIGURES

[0294] FIG. 1 The time-dependent titer of the expression of mAb-A in different cell lines: (1)=BAX/BAK-knock-out; (2)=ICAM-1-knock-out; (3)=control, no-knock-out; (4)=MYC-knock-out; (5)=BAX/BAK/ICAM-1/MYC/S1RT-1-knock-out; (6)=SIRT-1-knock-out.

[0295] FIG. 2 The time-dependent titer of the expression of mAb-B in different cell lines: (1)=BAX/BAK-knock-out; (2)=ICAM-1-knock-out; (3)=control, no-knock-out; (4)=MYC-knock-out; (5)=BAX/BAK/ICAM-1/MYC/S1RT-1-knock-out; (6)=SIRT-1-knock-out.

[0296] FIG. 3 The increase of average cell diameter over cultivation time for a cell expressing mAb-B: (1)=control, no-knock-out; (2)=MYC-knock-out; (3)=BAX/BAK/ICAM-1/MYC/S1RT-1-knock-out.

DESCRIPTION OF THE SEQUENCES

[0297] All gRNA sequences contain only the genome-targeting sequence of the gRNA.

TABLE-US-00004 SEQIDNO:01:exemplarysequenceofanL3 recombinaserecognitionsequence: AAGTCTCC SEQIDNO:02:exemplarysequenceofa2L recombinaserecognitionsequence: GCATACAT SEQIDNO:03:exemplarysequenceofaLoxFas recombinaserecognitionsequence: TACCTTTC SEQIDNO:04-06:exemplaryvariantsofhuman CMVpromoter: SEQIDNO:04: GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA TCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTAGCATGGTGATGCGGTTTTGGCAGTAC ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG GGAGGTCTATATAAGCAGAGCTCCGTTTAGTGAACGTCAG ATC SEQIDNO:05: GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA TCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTAGCATGGTGATGCGGTTTTGGCAGTAC ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG GGAGGTCTATATAAGCAGAGCTCCGTTTAGTGAACGTCAG ATCTAGCTCTGGGAGAGGAGCCCAGCACTAGAAGTCGGCG GTGTTTCCATTCGGTGATCAGCACTGAACACAGAGGAAGC TTGCCGCCACC SEQIDNO:06: CTGCAGTGAATAATAAAATGTGTGTTTGTCCGAAATACGC GTTTTGAGATTTCTGTCGCCGACTAAATTCATGTCGCGCGA TAGTGGTGTTTATCGCCGATAGAGATGGCGATATTGGAAA AATCGATATTTGAAAATATGGCATATTGAAAATGTCGCCG ATGTGAGTTTCTGTGTAACTGATATCGCCATTTTTCCAAAA GTGATTTTTGGGCATACGCGATATCTGGCGATAGCGCTTAT ATCGTTTACGGGGGATGGCGATAGACGACTTTGGTGACTT GGGCGATTCTGTGTGTCGCAAATATCGCAGTTTCGATATA GGTGACAGACGATATGAGGCTATATCGCCGATAGAGGCGA CATCAAGCTGGCACATGGCCAATGCATATCGATCTATACA TTGAATCAATATTGGCCATTAGCCATATTATTCATTGGTTA TATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGT TGTATCCATATCATAATATGTACATTTATATTGGCTCATGT CCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTA TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCC GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACG CCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGAT GCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCA ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC AAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG CTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC CTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCC GTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTG GCTTGGGGTCTATACACCCCCGCTTCCTCATGTTATAGGTG ATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTA TTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAA TCCATAACATGGCTCTTTGCCACAACTCTCTTTATTGGCTA TATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCT GTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATT CACATATACAACACCACCGTCCCCAGTGCCCGCAGTTTTTA TTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACG TGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTC TACATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGGT CGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACT TAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCAC AAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCG GGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAA GGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGT GTTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTG TTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTG CTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAA CAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTC CTTGACACGGTTTAAACGCCGCCACC SEQIDNO:07:exemplarySV40polyadenylation signalsequence: AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACCA TTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC ATGTCTG SEQIDNO:08:exemplarybGHpolyadenylation signalsequence: TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGC CTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA TGCGGTGGGCTCTATGG SEQIDNO:09:exemplaryhGTterminatorsequence: CAGGATAATATATGGTAGGGTTCATAGCCAGAGTAACCTT TTTTTTTAATTTTTATTTTATTTTATTTTTGAG SEQIDNO:10:exemplarySV40promotersequence: AGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGC AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAAC TCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAA TTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCT GAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCC TAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATT TTCG SEQIDNO:11:exemplaryGFPnucleicacid sequence: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGC CCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGC TGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTAC GGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGC AGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTAC AAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCC GGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCG ACGGTACCGCGGGCCCGGGATCCACCGGATCTAGATGA SEQIDNO:12-14:SIRT-1guideRNAs SEQIDNO:12: gRNA_SIRT1_1: TATCATCCAACTCAGGTGGA SEQIDNO:13: gRNA_SIRT1_2: GCAGCATCTCATGATTGGCA SEQIDNO:14: gRNA_SIRT1_3: GCATTCTTGAAGTAACTTCA SEQIDNO:15-16: SIRT-1PCRprimer SEQIDNO:15: SIRT1_for: ATGGCAGTTTTAGACACC SEQIDNO:16: SIRT1_rev: CTTGGAACTCAGACAAGG SEQIDNO:17-19:MYCguideRNAs SEQIDNO:17: gRNA_MYC_1: CTATGACCTCGACTACGACT SEQIDNO:18: gRNA_MYC_2: GGACGCAGCGACCGTCACAT SEQIDNO:19: gRNA_MYC_3: CACCATCTCCAGCTGATCCG SEQIDNO:20-21:MYCPCRprimer SEQIDNO:20: MYC_for: CACACACACACTTGGAAG SEQIDNO:21: MYC_rev: CTTGATGAAGGTCTCGTC SEQIDNO:22-24and35:ICAM-1guideRNAs SEQIDNO:22: gRNA_ICAM1_1: ACCTGCATGGATGCACCCCG SEQIDNO:23: gRNA_ICAM1_2: GCACCGTGCCCACCTCCAGG SEQIDNO:24: gRNA_ICAM1_3: TAACCGCCAGAGAAAGATC SEQIDNO:35: gRNA_ICAM1_4: ACCTGCATGGATGCACCCCG SEQIDNO:25-26:ICAM-1PCRprimer SEQIDNO:25: ICAM1_for: CCAAGCTAGATGATGTGAG SEQIDNO:26: ICAM1_rev: GCCCTACCCTTTTAATAC SEQIDNO:27-29and36-37:BAKguideRNAs SEQIDNO:27: gRNA_BAK_1: TACAGCATCTTGGGTCAGGT SEQIDNO:28: gRNA_BAK_2: GTCCATCTCGGGGTTGGCAG SEQIDNO:29: gRNA_BAK_3: AATCTTGGTGAAGAGTTCGT SEQIDNO:36: gRNA_BAK_4: TCATCACAGTCCTGCCTAGG SEQIDNO:37: gRNA_BAK_5: ATGGCGTCTGGACAAGGACC SEQIDNO:30-31:BAKPCRprimer SEQIDNO:30: BAK_for: CGTATCTGAGTTCACGAAC SEQIDNO:31: BAK_rev: CCATCAGGAACAAGAGAC SEQIDNO:32-34and38-39:BAXguideRNAs SEQIDNO:32: gRNA_BAX_1: ACAGGGGCCTTTTTGCTACA SEQIDNO:33: gRNA_BAX_2: GCTCATCTCCAATTCGCCTG SEQIDNO:34: gRNA_BAX_3: ACGAGAGGTCTTCTTCCGTG SEQIDNO:38: gRNA_BAX_4: GGGTCGGGGGAGCAGCTCGG SEQIDNO:39: gRNA_BAX_5: GGGTCCCGAAGTATGAGAGG SEQIDNO:35-36:BAXPCRprimer SEQIDNO:35: BAX_for: ATCTTGTCTCCCTCGTAG SEQIDNO:36: BAX_rev: TCCTGGACTTCTCTAACC

EXAMPLES

Example 1

General Techniques

[0298] 1) Recombinant DNA Techniques

[0299] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, (1989). The molecular biological reagents were used according to the manufacturer's instructions.

[0300] 2) DNA Sequence Determination

[0301] DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany) or Eurofins Genomics GmbH (Ebersberg, Germany) or Microsynth AG (Balgach, Switzerland).

[0302] 3) DNA and Protein Sequence Analysis and Sequence Data Management

[0303] The EMBOSS (European Molecular Biology Open Software Suite) software package and Geneious prime 2021 (Auckland, New Zealand) were used for sequence creation, mapping, analysis, annotation and illustration.

[0304] 4) Gene and Oligonucleotide Synthesis

[0305] Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany) or Twist Bioscience (San Francisco, USA). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0306] 5) Reagents

[0307] All commercial chemicals, antibodies and kits were used as provided according to the manufacturer's protocol if not stated otherwise.

[0308] 6) Cultivation of TI Host Cell Line

[0309] TI CHO host cells were cultivated at 37 C. in a humidified incubator with 85% humidity and 5% CO.sub.2. They were cultivated in a proprietary DMEM/F12-based medium containing 300 g/ml Hygromycin B and 4 g/ml of a second selection marker. The cells were splitted every 3 or 4 days at a concentration of 0.310E6 cells/ml in a total volume of 30 ml. For the cultivation 125 ml non-baffle Erlenmeyer shake flasks were used. Cells were shaken at 150 rpm with a shaking amplitude of 5 cm. The cell count was determined with Cedex HiRes Cell Counter (Roche). Cells were kept in culture until they reached an age of 60 days.

[0310] 7) Cloning

[0311] a) General:

[0312] Cloning with R-sites depends on DNA sequences next to the gene of interest (GOI) that are equal to sequences lying in following fragments. Like that, assembly of fragments is possible by overlap of the equal sequences and subsequent sealing of nicks in the assembled DNA by a DNA ligase. Therefore, a cloning of the single genes in particular preliminary vectors containing the right R-sites is necessary. After successful cloning of these preliminary vectors the gene of interest flanked by the R-sites is cut out via restriction digest by enzymes cutting directly next to the R-sites. The last step is the assembly of all DNA fragments in one-step. In more detail, a 5-exonuclease removes the 5-end of the overlapping regions (R-sites). After that, annealing of the R-sites can take place and a DNA polymerase extends the 3-end to fill the gaps in the sequence. Finally, the DNA ligase seals the nicks in between the nucleotides. Addition of an assembly master mix containing different enzymes like exonucleases, DNA polymerases and ligases, and subsequent incubation of the reaction mix at 50 C. leads to an assembly of the single fragments to one plasmid. After that, competent E. coli cells are transformed with the plasmid.

[0313] For some vectors, a cloning strategy via restriction enzymes was used. By selection of suitable restriction enzymes, the wanted gene of interest can be cut out and afterwards inserted into a different vector by ligation. Therefore, enzymes cutting in a multiple cloning site (MCS) are preferably used and chosen in a smart manner, so that a ligation of the fragments in the correct array can be conducted. If vector and fragment are previously cut with the same restriction enzyme, the sticky ends of fragment and vector fit perfectly together and can be ligated by a DNA ligase, subsequently. After ligation, competent E. coli cells are transformed with the newly generated plasmid.

[0314] b) Cloning Via Restriction Digestion:

[0315] For the digest of plasmids with restriction enzymes, the following components were pipetted together on ice:

TABLE-US-00005 TABLE Restriction Digestion Reaction Mix component ng (set point) l purified DNA tbd tbd CutSmart Buffer (10) 5 Restriction Enzyme 1 PCR-grade Water ad 50 Total 50

[0316] If more enzymes were used in one digestion, 111.1 of each enzyme was used and the volume adjusted by addition of more or less PCR-grade water. All enzymes were selected on the preconditions that they are qualified for the use with CutSmart buffer from new England Biolabs (100% activity) and have the same incubation temperature (all 37 C.).

[0317] Incubation was performed using thermomixers or thermal cyclers, allowing incubating the samples at a constant temperature (37 C.). During incubation the samples were not agitated. Incubation time was set at 60 min. Afterwards the samples were directly mixed with loading dye and loaded onto an agarose electrophoresis gel or stored at 4 C./on ice for further use.

[0318] A 1% agarose gel was prepared for gel electrophoresis. Therefor 1.5 g of multi-purpose agarose were weighed into a 125 Erlenmeyer shake flask and filled up with 150 ml TAE-buffer. The mixture was heated up in a microwave oven until the agarose was completely dissolved. 0.5 g/ml ethidium bromide were added into the agarose solution. Thereafter the gel was cast in a mold. After the agarose was set, the mold was placed into the electrophoresis chamber and the chamber filled with TAE-buffer. Afterwards the samples were loaded. In the first pocket (from the left), an appropriate DNA molecular weight marker was loaded, followed by the samples. The gel was run for around 60 minutes at <130 V. After electrophoresis, the gel was removed from the chamber and analyzed in an UV-Imager.

[0319] The target bands were cut and transferred to 1.5 ml Eppendorf tubes. For purification of the gel, the QIAquick Gel Extraction Kit from Qiagen was used according to the manufacturer's instructions. The DNA fragments were stored at 20 C. for further use.

[0320] The fragments for the ligation were pipetted together in a molar ratio of 1:2, 1:3 or 1:5 vector to insert, depending on the length of the inserts and the vector-fragments and their correlation to each other. If the fragment, that should be inserted into the vector was short, a 1:5-ratio was used. If the insert was longer, a smaller amount of it was used in correlation to the vector. An amount of 50 ng of vector were used in each ligation and the particular amount of insert calculated with NEBioCalculator. For ligation, the T4 DNA ligation kit from NEB was used. An example for the ligation mixture is depicted in the following Table.

TABLE-US-00006 TABLE Ligation Reaction Mix component ng (set point) conc. [ng/l] l T4 DNA Ligase Buffer 2 (10) 50 50 1 Vector DNA (4000 bp) 125 20 6.25 Insert DNA (2000 bp) 9.75 Nuclease-free Water 1 T4 Ligase Total 20

[0321] All components were pipetted together on ice, starting with the mixing of DNA and water, addition of buffer and finally addition of the enzyme. The reaction was gently mixed by pipetting up and down, briefly microfuged and then incubated at room temperature for 10 minutes. After incubation, the T4 ligase was heat inactivated at 65 C. for 10 minutes. The sample was chilled on ice. In a final step, 10-beta competent E. coli cells were transformed with 2 l of the ligated plasmid (see below).

[0322] c) Cloning Via R-Site Assembly:

[0323] For assembly, all DNA fragments with the R-sites at each end were pipetted together on ice. An equimolar ratio (0.05 ng) of all fragments was used, as recommended by the manufacturer, when more than 4 fragments are being assembled. One-half of the reaction mix was embodied by NEBuilder HiFi DNA Assembly Master Mix. The total reaction volume was 40 l and was reached by a fill-up with PCR-clean water. In the following Table, an exemplary pipetting scheme is depicted.

TABLE-US-00007 TABLE Assembly Reaction Mix pmol ng conc. component bp (set point) (set point) [ng/l] l Insert 1 2800 0.05 88.9 21 4.23 Insert 2 2900 0.05 90.5 35 2.59 Insert 3 4200 0.05 131.6 35.5 3.71 Insert 4 3600 0.05 110.7 23 4.81 Vector 4100 0.05 127.5 57.7 2.21 NEBuilder HiFi DNA 20 Assembly Master Mix PCR-clean Water 2.45 Total 40

[0324] After set up of the reaction mixture, the tube was incubated in a thermocycler at constantly C. for 60 minutes. After successful assembly, 10-beta competent E. coli bacteria were transformed with 211.1 of the assembled plasmid DNA (see below).

[0325] d) Transformation 10-Beta Competent E. coli Cells:

[0326] For transformation, the 10-beta competent E. coli cells were thawed on ice. After that, 211.1 of plasmid DNA were pipetted directly into the cell suspension. The tube was flicked and put on ice for 30 minutes. Thereafter, the cells were placed into the 42 C.-warm thermal block and heat-shocked for exactly 30 seconds. Directly afterwards, the cells were chilled on ice for 2 minutes. 950 l of NEB 10-beta outgrowth medium were added to the cell suspension. The cells were incubated under shaking at 37 C. for one hour. Then, 50-100 l were pipetted onto a pre-warmed (37 C.) LB-Amp agar plate and spread with a disposable spatula. The plate was incubated overnight at 37 C. Only bacteria, which have successfully incorporated the plasmid, carrying the resistance gene against ampicillin, can grow on these plates. Single colonies were picked the next day and cultured in LB-Amp medium for subsequent plasmid preparation.

[0327] e) Bacterial Culture:

[0328] Cultivation of E. coli was done in LB-medium, short for Luria Bertani, which was spiked with 1 ml/L 100 mg/ml ampicillin resulting in an ampicillin concentration of 0.1 mg/ml. For the different plasmid preparation quantities, the following amounts were inoculated with a single bacterial colony.

TABLE-US-00008 TABLE E. coli cultivation volumes volume LB-Amp incubation time quantity plasmid preparation medium [ml] [h] Mini-Prep 96-well (EpMotion) 1.5 23 Mini-Prep 15 ml-tube 3.6 23 Maxi-Prep 200 16

[0329] For Mini-Prep, a 96-well 2 ml deep-well plate was filled with 1.5 ml LB-Amp medium per well. The colonies were picked and the toothpick was tuck in the medium. When all colonies were picked, the plate closed with a sticky air porous membrane. The plate was incubated in a 37 C. incubator at a shaking rate of 200 rpm for 23 hours.

[0330] For Mini-Preps a 15 ml-tube (with a ventilated lid) was filled with 3.6 ml LB-Amp medium and equally inoculated with a bacterial colony. The toothpick was not removed but left in the tube during incubation. Like the 96-well plate, the tubes were incubated at 37 C., 200 rpm for 23 hours.

[0331] For Maxi-Prep 200 ml of LB-Amp medium were filled into an autoclaved glass 1 L Erlenmeyer flask and inoculated with 1 ml of bacterial day-culture, which was roundabout hours old. The Erlenmeyer flask was closed with a paper plug and incubated at 37 C., 200 rpm for 16 hours.

[0332] f) Plasmid Preparation:

[0333] For Mini-Prep, 50 l of bacterial suspension were transferred into a 1 ml deep-well plate. After that, the bacterial cells were centrifuged down in the plate at 3000 rpm, 4 C. for 5 min. The supernatant was removed and the plate with the bacteria pellets placed into an EpMotion. After approx. 90 minutes, the run was done and the eluted plasmid-DNA could be removed from the EpMotion for further use.

[0334] For Mini-Prep, the 15 ml tubes were taken out of the incubator and the 3.6 ml bacterial culture splitted into two 2 ml Eppendorf tubes. The tubes were centrifuged at 6,800g in a tabletop microcentrifuge for 3 minutes at room temperature. After that, Mini-Prep was performed with the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. The plasmid DNA concentration was measured with Nanodrop.

[0335] Maxi-Prep was performed using the Macherey-Nagel NucleoBond Xtra Maxi EF Kit according to the manufacturer's instructions. The DNA concentration was measured with Nanodrop.

[0336] g) Ethanol precipitation:

[0337] The volume of the DNA solution was mixed with the 2.5-fold volume ethanol 100%. The mixture was incubated at 20 C. for 10 min. Then the DNA was centrifuged for 30 min. at 14,000 rpm, 4 C. The supernatant was carefully removed and the pellet washed with 70% ethanol. Again, the tube was centrifuged for 5 min. at 14,000 rpm, 4 C. The supernatant was carefully removed by pipetting and the pellet dried. When the ethanol was evaporated, an appropriate amount of endotoxin-free water was added. The DNA was given time to re-dissolve in the water overnight at 4 C. A small aliquot was taken and the DNA concentration was measured with a Nanodrop device.

Example 2

[0338] Plasmid Generation

[0339] Expression Cassette Composition

[0340] For the expression of an antibody chain, a transcription unit comprising the following functional elements were used: [0341] the immediate early enhancer and promoter from the human cytomegalovirus including intron A, [0342] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0343] a murine immunoglobulin heavy chain signal sequence, [0344] a nucleic acid encoding the respective antibody chain, [0345] the bovine growth hormone polyadenylation sequence (BGH pA), and [0346] optionally the human gastrin terminator (hGT).

[0347] Beside the expression unit/cassette including the desired gene to be expressed, the basic/standard mammalian expression plasmid contains: [0348] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and [0349] a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0350] Front- and Back-Vector Cloning

[0351] To construct two-plasmid antibody constructs, antibody HC and LC fragments were cloned into a front vector backbone containing L3 and LoxFas sequences, and a back vector containing LoxFas and 2L sequences and a pac selectable marker. The Cre recombinase plasmid pOG231 (Wong, E. T., et al., Nucl. Acids Res. 33 (2005) e147; O'Gorman, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) was used for all RMCE processes.

[0352] The cDNAs encoding the respective antibody chains were generated by gene synthesis (Geneart, Life Technologies Inc.). The gene synthesis and the backbone-vectors were digested with HindIII-RF and EcoRI-HF (NEB) at 37 C. for 1 h and separated by agarose gel electrophoresis. The DNA-fragment of the insert and backbone were cut out from the agarose gel and extracted by QIAquick Gel Extraction Kit (Qiagen). The purified insert and backbone fragment was ligated via the Rapid Ligation Kit (Roche) following the manufacturer's protocol with an Insert/Backbone ratio of 3:1. The ligation approach was then transformed in competent E. coli DH5a via heat shock for 30 sec. at 42 C. and incubated for 1 h at 37 C. before they were plated out on agar plates with ampicillin for selection. Plates were incubated at 37 C. overnight.

[0353] On the following day clones were picked and incubated overnight at 37 C. under shaking for the Mini or Maxi-Preparation, which was performed with the EpMotion 5075 (Eppendorf) or with the QIAprep Spin Mini-Prep Kit (Qiagen)/NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure the absence of any undesirable mutations (Sequi Serve GmbH).

[0354] In the second cloning step, the previously cloned vectors were digested with KpnI-HF/SalI-HF and SalI-HF/MfeI-HF with the same conditions as for the first cloning. The TI backbone vector was digested with KpnI-HF and MfeI-HF. Separation and extraction was performed as described above. Ligation of the purified insert and backbone was performed using T4 DNA Ligase (NEB) following the manufacturing protocol with an Insert/Insert/Backbone ratio of 1:1:1 overnight at 4 C. and inactivated at 65 C. for 10 min. The following cloning steps were performed as described above.

[0355] The cloned plasmids were used for the TI transfection and pool generation.

Example 3

[0356] Cultivation, Transfection, Selection and Single Cell Cloning

[0357] TI host cells were propagated in disposable 125 ml vented shake flasks under standard humidified conditions (95% rH, 37 C., and 5% CO.sub.2) at a constant agitation rate of 150 rpm in a proprietary DMEM/F12-based medium. Every 3-4 days the cells were seeded in chemically defined medium containing selection marker 1 and selection marker 2 in effective concentrations with a concentration of 310E5 cells/ml. Density and viability of the cultures were measured with a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[0358] For stable transfection, equimolar amounts of front and back vector were mixed. Total DNA used per transfection was 30 g with plasmid ratio 2.5:2.5:1 (front-, back-, Cre plasmid).

[0359] Two days prior to transfection TI host cells were seeded in fresh medium with a density of 410E5 cells/ml. Transfection was performed with the MaxCyte STX electroporation device (MaxCyte Inc., Gaithersburg) using OC-400 electroporation cassettes according to the manufacturer's protocol. 310E7 cells were transfected with a total of 30 g nucleic acids, i.e. either with 30 g plasmid (with a molar ratio of 2.5:2.5:1 of front:back:Cre plasmid)) or with 5 g Cre mRNA and 25 g front-and back-vector mixture. After transfection, the cells were seeded in 30 ml medium without selection agents.

[0360] On day 5 after seeding the cells were centrifuged and transferred to 80 mL chemically defined medium containing puromycin (selection agent 1) and 1-(2-deoxy-2-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) at effective concentrations at 610E5 cells/ml for selection of recombinant cells. The cells were incubated at 37 C., 150 rpm. 5% CO2, and 85% humidity from this day on without splitting. Cell density and viability of the culture was monitored regularly. When the viability of the culture started to increase again, the concentrations of selection agents 1 and 2 were reduced to about half the amount used before.

[0361] In more detail, to promote the recovering of the cells, the selection pressure was reduced if the viability is >40% and the viable cell density (VCD) is >0.510E6 cells/mL. Therefore, 410E5 cells/ml were centrifuged and resuspended in 40 ml selection media II (chemically defined medium, selection marker 1 & 2). The cells were incubated with the same conditions as before and also not split.

[0362] Ten days after starting selection, the success of Cre mediated cassette exchange was checked by flow cytometry measuring the expression of intracellular GFP and extracellular heterologous polypeptide bound to the cell surface. An APC antibody (allophycocyanin-labeled F(ab)2 Fragment goat anti-human IgG) against human antibody light and heavy chain was used for FACS staining. Flow cytometry was performed with a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). Ten thousand events per sample were measured. Living cells were gated in a plot of forward scatter (FSC) against side scatter (SSC). The live cell gate was defined with non-transfected TI host cells and applied to all samples by employing the FlowJo 10.8.1 EN software (TreeStar, Olten, Switzerland). Fluorescence of GFP was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). Heterologous polypeptide was measured in the APC channel (excitation at 645 nm, detection at 660 nm). Parental CHO cells, i.e., those cells used for the generation of the TI host cell, were used as a negative control with regard to GFP and heterologous polypeptide expression. Fourteen to twenty-one days after the selection had been started, the viability exceeded 90% and selection was considered as complete.

[0363] After selection, the pool of stably transfected cells can be subjected to single-cell cloning by limiting dilution. For this purpose, cells are stained with Cell Tracker Green (Thermo Fisher Scientific, Waltham, MA) and plated in 384-well plates with 0.6 cells/well. For single-cell cloning and all further cultivation steps, selection agent 2 is omitted from the medium. Wells containing only one cell are identified by bright field and fluorescence-based plate imaging. Only wells that contain one cell are further considered. Approximately three weeks after plating colonies are picked from confluent wells and further cultivated in 96-well plates.

Example 4

[0364] FACS Screening

[0365] FACS analysis was performed to check the transfection efficiency and the RMCE efficiency of the transfection. 410E5 cells of the transfected approaches were centrifuged (1200 rpm, 4 min.) and washed twice with 1 mL PBS. After the washing steps with PBS the pellet was resuspended in 400 L PBS and transferred in FACS tubes (Falcon Round-Bottom Tubes with cell strainer cap; Corning). The measurement was performed with a FACS Canto II and the data were analyzed by the software FlowJo.

Example 5

[0366] Fed-Batch Cultivation

[0367] Fed-batch production cultures were performed in shake flasks or Ambr 15 vessels (Sartorius Stedim) with proprietary chemically defined medium. Cells were seeded at 210E6 cells/ml on day 0. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell count (VCC) and percent viability of cells in culture was measured on days 0, 3, 7, 10, and 14 using a Cedex HiRes instrument (Roche Diagnostics GmbH, Mannheim, Germany). Glucose, lactate and product titer concentrations were measured on days 3, 5, 7, 10, 12 and 14 using a Cobas Analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The supernatant was harvested 14 days after start of fed-batch cultivation by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and cleared by filtration (0.22 m). Day 14 titers were determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

Example 6

[0368] RNP-Based CRISPR-Cas9 Gene Knockouts in CHO Cells

[0369] Material/Resources: [0370] Geneious 2021.2.2 software for guide and primer design [0371] CHO TI host cell line; cultivation state: day 30-60 [0372] Gibco TrueCut Cas9 Protein, A45220P, Thermo Fisher [0373] sgRNA (custom designed against target gene, 3 nm chemically modified sgRNA, Synthego) [0374] medium (200 g/ml Hygromycin B, 4 g/ml selection agent 2) [0375] DPBSDulbecco's Phosphate-Buffered Saline w/o Ca and Mg (Thermo Fisher) [0376] Microplate 24 deep well plate (Agilent Technologies, Porvoir science) with cover (self-made) [0377] Thin, long RNase, DNase, pyrogen free filter tips for loading OC-100 cassettes. (Biozyme) [0378] Hera Safe Hood (Thermo Fisher) [0379] Cedex HiRes Analyzer (Innovatis) [0380] Liconic Incubator Storex IC [0381] HyClone electroporation buffer [0382] MaxCyte OC-100 cassettes [0383] MaxCyte STX electroporation system [0384] CRISPR-Cas9 RNP delivery

[0385] RNPs were preassembled by mixing 30 pmol Cas9 with 30 pmol g gRNA mix (equal ratio of each gRNAsee Table below for exemplary genes-specific gRNA sequences) and incubated for 20 minutes at RT. Cells with a concentration between 2-410E6 cell/mL were centrifuged (3 minutes, 300 g). Afterwards the cells were resuspended in 90 L HyClone electroporation buffer. The pre-incubated RNP mix was added to the cells and incubated for 5 minutes. The cell/RNP solution was then transferred into an OC-100 cuvette and electroporated with program CHO2 using a MaxCyte electroporation system. Immediately after electroporation, the cell suspension was transferred into a 24 dwell and incubated at 37 C. for 30 minutes. Fresh and pre-warmed medium was added to result in a final cell concentration of 110E6 and incubated at 37 C. with shaking at 350 rpm for cell expansion. For genomic DNA preparation (day 6 or 8), QuickExtract kit (Lucigen) was added to the cells and served as a PCR template. Specific gene amplicons were PCR-amplified using standard Q5 Hot Start Polymerase protocol (NEB) and gene-specific primers that span the gRNA target sites (see Table below for examples). The respective amplicon was purified using QIAquick PCR purification kit (Qiagen) and analyzed by Sanger sequencing by Eurofins Genomics GmbH to verify gene inactivation by knockout.

Example 7

[0386] Fed-Batch Cultivation

[0387] Fed-batch production cultures were performed in Ambr 15 or Ambr 250 vessels (Sartorius Stedim) with proprietary chemically defined medium. Cells were seeded at 210E6 cells/ml. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell count (VCC) and percent viability of cells in culture was measured on days 0, 3, 7, 10, 12 and 14 using a Cedex HiRes (Roche Diagnostics GmbH, Mannheim, Germany). Glucose concentration, lactate concentration and product titer were measured on days 3, 5, 7, 10, 12 and 14 using a Cobas analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The supernatant was harvested 10, 12 or 14 days after start of fed-batch by centrifugation (10 min., 1000 rpm followed by 10 min., 4000 rpm) and cleared by filtration (0.22 m). Harvest titers were further determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

Example 8

[0388] High Cell Density Fed-Batch Cultivation

[0389] Fed-batch production cultures were performed in Ambr 15 or Ambr 250 vessels (Sartorius Stedim) with proprietary chemically defined medium. Cells were seeded at 1510E6 cells/ml on day 0. Cultures received proprietary feed medium on days 1, 3, and 6. Viable cell count (VCC) and percent viability of cells in culture was measured on days 0, 3, 7, 10, 12 and 14 using a Cedex HiRes instrument (Roche Diagnostics GmbH, Mannheim, Germany). Glucose concentration, lactate concentration and product titer were measured on days 3, 5, 7, 10, 12, and 14 using a Cobas Analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The supernatant was harvested 10 or 12 or 14 days after start of the cultivation by centrifugation (10 min., 1000 rpm followed by 10 min., 4000 rpm) and cleared by filtration (0.22 m). Harvest titers were further determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).