IP Library › Granted Patent US 12,590,880
Granted Patent B2
US 12,590,880 · App. 18/181,803 · Granted Mar 31, 2026

Flare (flow cytometry attenuated reporter expression) technology for rapid bulk sorting

Inventors: Victor R. Cairns (Cambridge, MA); Jose Ignacio Sancho Chavida (Cambridge, MA); Christine DeMaria (Cambridge, MA)
Assignee: Genzyme Corporation
G01N15/14C07K16/00C12N15/1086C12N15/85G01N33/5044C07K2317/10C12N2510/02G01N15/149G01N2333/70592
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Quick Facts
Patent No.
US 12,590,880
App. No.
18/181,803
Granted
Mar 31, 2026
Kind
B2
Abstract

Provided herein are methods and compositions for batch production of producer cells using fluorescence activated cell sorting (FACS). In some aspects, the disclosure provides a drug-selection-free method for batch production of producer cells using FACS. Such batch production methods and compositions can be further utilized to generate clonal populations of producer cells, e.g., for large-scale manufacturing of a polypeptide of interest.

Claims (31)

1 . A method of increasing viability of cells after fluorescence activated cell sorting (FACS), the method comprising:

using a first round of FACS to select based on a viability marker a first sub-population of cells from a population of cells expressing a target polypeptide, and

using a second round of FACS to select based on the viability marker a second sub-population of cells from the first sub-population of cells,

wherein the first round and second round of FACS occur within 8 hours of each other, and wherein the viability marker is a forward scatter/side scatter population differentiation and/or a propidium iodide stain.

2 . The method of claim 1 , wherein the second sub-population has a post sort viability improvement of 1.2- to 4-fold compared to the initial sub-population post sort viability.

3 . The method of claim 1 , further comprising expanding the second sub-population.

4 . The method of claim 1 , further comprising individually culturing cells from the second sub-population in order to generate one or more clonal populations.

5 . The method of claim 1 , wherein the increasing viability of cells after FACS is performed after a batch selection method.

6 . The method of claim 5 , wherein the batch selection method comprises:

(a) providing a heterogeneous population of producer cells, wherein the producer cells in the population express varying levels of a FACS selectable polypeptide and the target polypeptide that are encoded by the same multicistronic mRNA,

(b) selecting from the heterogeneous population of producer cells a first heterogeneous sub-population of producer cells using FACS, wherein the producer cells in the first heterogeneous sub-population express the FACS selectable polypeptide at a level that is higher than the level of at least 80% of the producer cells in the heterogeneous population in (a), and

(c) expanding the first heterogeneous sub-population of producer cells in drug-selection-free medium, thereby producing an expanded first heterogeneous sub-population of producer cells.

7 . The method of claim 6 , wherein the selection in step (b) is performed between 2 to about 15 days after transfection of the heterogeneous population of producer cells.

8 . The method of claim 6 , wherein the selection in step (b) is performed two days after transfection.

9 . The method of claim 6 , wherein the selection in step (b) is performed three days after transfection.

10 . The method of claim 6 , wherein the heterogeneous population of producer cells in (a) is produced by transfecting cells with a vector that encodes the multicistronic mRNA and subjecting the transfected cells to one round of medium-based selection to select cells expressing varying levels of the multicistronic mRNA.

11 . The method of claim 10 , wherein the vector further contains a dihydrofolate reductase (DHFR) gene.

12 . The method of claim 10 , wherein the medium-based selection is methotrexate-containing medium or nucleotide-deficient medium.

13 . The method of claim 6 , wherein the drug-selection-free medium is methotrexate-free medium or methionine sulphoximine-free medium.

14 . The method of claim 6 , further comprising:

(d) selecting from the expanded first heterogeneous sub-population of producer cells in (c) a second heterogeneous sub-population of producer cells using FACS, wherein the producer cells in the second sub-population express the FACS selectable polypeptide at a level that is higher than the level of at least 80 % of the producer cells in the expanded first heterogeneous sub-population of producer cells in (c), and

(e) expanding the second heterogeneous sub-population of producer cells in a drug-selection-free medium, thereby producing an expanded second heterogeneous sub-population of producer cells.

15 . The method of claim 6 , wherein the multicistronic mRNA comprises a first open reading frame (ORF) that encodes the FACS selectable polypeptide and a second ORF that encodes the target polypeptide, wherein the first ORF is 5′ to the second ORF.

16 . The method of claim 15 , wherein the first ORF has a non-AUG start codon.

17 . The method of claim 15 , wherein the second ORF has an AUG start codon.

18 . The method of claim 15 , wherein the ORF that encodes the FACS selectable polypeptide is devoid of any AUG sequences.

19 . The method of claim 6 , wherein the target polypeptide is selected from the group consisting of therapeutic agents, secreted proteins, antibodies, and Fc fusion proteins.

20 . The method of claim 1 , wherein the second round of FACS occurs as an immediate sort after the first round of FACs without any intervening cell steps between the first and second round of FACS.

21 . The method of claim 1 , wherein the first round and second round of FACS occur within 30 minutes to within 8 hours of each other.

22 . The method of claim 1 , wherein the first round and second round of FACS occur within 30 minutes of each other.

23 . The method of claim 1 , wherein the first round and second round of FACS occur within 60 minutes of each other.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 2, 2025
From: CAIRNS, VICTOR R.; CHAVIDA, JOSE IGNACIO SANCHO; DEMARIA, CHRISTINE
To: GENZYME CORPORATION
Reel/Frame 071597/0206 →
Continuity (3)
Division 15288036 · Oct 7, 2016
Provisional Application 62239515 · Oct 9, 2015
Related Publication 20230304913A1 · Sep 28, 2023
References Cited (169)
US 4812405A · Lair et al. · 1989 [cited by applicant]
US 4818700A · Cregg et al. · 1989 [cited by applicant]
US 4929555A · Cregg et al. · 1990 [cited by applicant]
US 4937190A · Palmenberg et al. · 1990 [cited by applicant]
US 5429746A · Shadle et al. · 1995 [cited by applicant]
US 5648267A · Reff · 1997 [cited by applicant]
US 5736383A · Raymond · 1998 [cited by applicant]
US 5888768A · Raymond · 1999 [cited by applicant]
US 5955349A · Raymond · 1999 [cited by applicant]
US 6258559B1 · Zamost · 2001 [cited by applicant]
US 6653132B1 · Keshet et al. · 2003 [cited by applicant]
US 6833254B2 · Dasgupta et al. · 2004 [cited by applicant]
US 7344886B2 · Enenkel et al. · 2008 [cited by applicant]
US 7759119B2 · Allbritton et al. · 2010 [cited by applicant]
US 7776584B2 · Richmond et al. · 2010 [cited by applicant]
US 8034612B2 · Richmond et al. · 2011 [cited by applicant]
US 8034625B2 · Richmond et al. · 2011 [cited by applicant]
US 8293520B2 · Richmond et al. · 2012 [cited by applicant]
US 8293525B2 · Richmond et al. · 2012 [cited by applicant]
US 8293526B2 · Richmond et al. · 2012 [cited by applicant]
US 8293527B2 · Richmond et al. · 2012 [cited by applicant]
US 10317329B2 · Cairns et al. · 2019 [cited by applicant]
US 11635363B2 · Cairns et al. · 2023 [cited by applicant]
US 11685943B2 · Cairns et al. · 2023 [cited by applicant]
US 20030022196A1 · Lorens et al. · 2003 [cited by applicant]
US 20040006216A1 · Waldmann · 2004 [cited by applicant]
US 20040082034A1 · Lee et al. · 2004 [cited by applicant]
US 20040148647A1 · Enenkel et al. · 2004 [cited by applicant]
US 20040180378A1 · Tozer et al. · 2004 [cited by applicant]
US 20050005310A1 · Chisolm et al. · 2005 [cited by applicant]
US 20050014150A1 · Atabekov et al. · 2005 [cited by applicant]
US 20050059004A1 · Atabekov et al. · 2005 [cited by applicant]
US 20050106580A1 · Enenkel et al. · 2005 [cited by applicant]
US 20050250107A1 · Di Colandrea et al. · 2005 [cited by applicant]
US 20060141577A1 · Otte et al. · 2006 [cited by applicant]
US 20060172382A1 · Otte et al. · 2006 [cited by applicant]
US 20060188964A1 · Mancia et al. · 2006 [cited by applicant]
US 20070037254A1 · Chisolm et al. · 2007 [cited by applicant]
US 20070238122A1 · Allbritton et al. · 2007 [cited by applicant]
US 20080248468A1 · Kubbies et al. · 2008 [cited by applicant]
US 20090239235A1 · Demaria et al. · 2009 [cited by applicant]
US 20100159030A1 · Kavallaris et al. · 2010 [cited by applicant]
US 20130090259A1 · Kotsopoulou et al. · 2013 [cited by applicant]
US 20130259923A1 · Bancel et al. · 2013 [cited by applicant]
US 20140073007A1 · Schuler et al. · 2014 [cited by applicant]
US 20160177318A1 · Assaraf et al. · 2016 [cited by applicant]
US 20170121734A1 · Cairns et al. · 2017 [cited by applicant]
US 20170227441A1 · Cairns et al. · 2017 [cited by applicant]
US 20180119192A1 · Cairns et al. · 2018 [cited by applicant]
US 20200018682A1 · Cairns et al. · 2020 [cited by applicant]
US 20200299743A1 · Cairns et al. · 2020 [cited by applicant]
US 20240026410A1 · Cairns et al. · 2024 [cited by applicant]
AR 062916A1 · 2008 [cited by applicant]
CN 108368504A · 2018 [cited by applicant]
EP 1749538A1 · 2007 [cited by applicant]
EP 2064337A2 · 2009 [cited by applicant]
EP 3359665A2 · 2018 [cited by applicant]
EP 3359666A1 · 2018 [cited by applicant]
EP 3523440A1 · 2019 [cited by applicant]
WO WO1991001374A1 · 1991 [cited by applicant]
WO WO1994026087A2 · 1994 [cited by applicant]
WO WO1995027071A2 · 1995 [cited by applicant]
WO WO2001004306A1 · 2001 [cited by applicant]
WO WO2001057212A1 · 2001 [cited by applicant]
WO WO2003099996A2 · 2003 [cited by applicant]
WO WO2004009823A1 · 2004 [cited by applicant]
WO WO2004060910A2 · 2004 [cited by applicant]
WO WO2005000888A2 · 2005 [cited by applicant]
WO WO2005094886A1 · 2005 [cited by applicant]
WO WO2008036255A2 · 2008 [cited by applicant]
WO WO2012001073A2 · 2012 [cited by applicant]
WO WO2014141037A1 · 2014 [cited by applicant]
WO WO2017062722A2 · 2017 [cited by applicant]
WO WO2017062724A1 · 2017 [cited by applicant]
Okumura, Takeshi, et al. “Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry.” Journal of bioscience and bioengineering 120.3 (2015): 340-346. (Year:… [cited by examiner]
Mancia, Filippo, et al. “Optimization of protein production in mammalian cells with a coexpressed fluorescent marker.” Structure 12.8 (2004): 1355-1360. (Year: 2004). [cited by examiner]
Yim, Sung Sun, et al. “Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS).” PLoS one 9.10 (2014): e108225. (Year: 2014). [cited by examiner]
Vitelli, Michael, et al. “Applications of flow cytometry sorting in the pharmaceutical industry: A review.” Biotechnology Progress 37.4 (2021): e3146. (Year: 2021). [cited by examiner]
Bailey et al. (2002) “High-Throughput Clonal Selectio of Recombinant CHO Cells Using a Dominant Selectable and Amplifiable Metallothionein-GFP Fusion Protein,” Biotech. Bioeng. 80(6):670-676. [cited by applicant]
Barka et al. (2004) “Production of Cell Lines Secreting TAT Fusion Proteins,” J. Histochem. and Cytochem., 52(4):469-477. [cited by applicant]
Barnes et al. (2003) “Stability of Protein Production from Recombinant Mammalian Cells,” Biotech. Bioeng. 81(6):631-639. [cited by applicant]
Bohm et al. (2004) “Screening for Improved Cell Performance: Selection of Subelones with Altered Production Kinetics or Improved Stability by Cell Sorting,” Biotechnol. Bioeng. 88(6):699-706. [cited by applicant]
Borth et al. (2000) “Efficient Selection of High-Producing Subelones During Gene Amplification of Recombinant Chinese Hamster Ovary Cells by Flow Cytometry and Cell Sorting,” Biotechnol. Bioeng. 71(4):266-273. [cited by applicant]
Brezinsky et al. (2003) “A Simple Method for Enriching Populations of Transfected CHO Cells for Cells of Higher Specific Productivity,” J. Immunol. Methods 277:141-155. [cited by applicant]
Browne et al. (2007) “Selection methods for high-producing mammalian cell lines,” Trends in Biotechnology. 25(9):425-432. [cited by applicant]
Cairns et al. (2011) “Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines,” Biotechnol. Bioeng. 108(11):2611-2622. [cited by applicant]
Carroll et al. (1993) “Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon,” J. Virol. 67(3):1433-1440. [cited by applicant]
Carroll et al. (2004) “The Selection of High-Producing Cell Lines Using Flow Cytometry and Cell Sorting”, Expi. Opin. Biol. Ther. 4(11):1821-1829. [cited by applicant]
Chen et al. (2004) “Highly Efficient Selection of the Stable Clones Expressing Antibody-IL-2 Fusion Protein by a Dicistronic Expression Vector Containing a Mutant Neo Gene,” J. Immunol. Methods. 295:49-56. [cited by applicant]
Chilov et al. (2004) “Toward construction of a self-sustained clock-like expression system based on the mammalian circadian clock,” Biotechnology and Bioengineering. 87(2):234-242. [cited by applicant]
Choe et al. (2005) “A Dual-Fluorescence Reporter System for High-Throughput Clone Characterization and Selection by Cell Sorting,” Nucleic Acids Res. 33(5):1-7. [cited by applicant]
Clontech Laboratories, Inc. (Sep. 1, 2005) “pIRES Vector Information,” Protocol No. PT3266-5. Version No. PR59976. [cited by applicant]
Condon et al. (2003) “Development of A Chinese Hamster Ovary Cell Line for Recombinant Adenovirus-Mediated Gene Expression,” Biotechnol. Prog. 19(1):137-143. [cited by applicant]
Davies et al. (1992) “The Sequence Context of the Initiation Codon in the Encephalomyocarditis Virus Leader Modulates Efficiency of Internal Translation Initiation,” J. Virology 66(4):1924-1932. [cited by applicant]
DeMaria (2012) “Selection of High Expressing Mammalian Cells by Surface Display of Reporters,” Ch. 3 In; Methods in Molecular Biology. 801:27-39. [cited by applicant]
DeMaria et al. (2007) “Accelerated Clone Selection for Recombinant CHO Cells Using a FACS-Based High-Throughput Screen,” Biotechnology Progress. 23(2):465-472. [cited by applicant]
Domagala, et al., “CD52 Antigen—A Review”, Medical Science Monitor, 2001, 7(2):325-331. [cited by applicant]
Dorn et al. (1990) “Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins,” Journal of Virology. 64(4):1616-1624. [cited by applicant]
Drapeau et al. (1994) “Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process,” Cytotechnology. 15:103-109. [cited by applicant]
Edwards et al. (2004) “Flow Cytometry For High-Throughput, I ligh Content Screening”, Curr. Opin. Chem. Biol. 8:392-398. [cited by applicant]
Examination Report corresponding to European Patent Application No. 07838397.3, dated Jul. 22, 2009. [cited by applicant]
Examination Report corresponding to European Patent Application No. 07838397.3, dated Jul. 7, 2011. [cited by applicant]
Fan, “Plasmid 101-Multicistronic Vectors”, AddGene, Webpage, Sep. 9, 2014. [cited by applicant]
Fletcher et al. (1992) “New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay acc… [cited by applicant]
Fux et al. (2004) “New-generation multicistronic expression platform: pTrident vectors containing size-optimized Ires elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors,” Biote… [cited by applicant]
Gaines et al. (1999) “pIRES-CD4t, a Dicistronic Expression Vector for MACS- or FACS-Based Selection of Transfected Cells,” BioTechniques. 26(4):683-688. [cited by applicant]
Genbank Database [Online] (Jan. 30, 2011) “ [cited by applicant]
Gupta et al. (1988) “ACG, the initiator codon for a Sendai virus protein,” Journal of Biological Chemistry. 263 (18):8553-8556. [cited by applicant]
Gurtu et al. (1996) “IRES Bicistronic Expression Vectors for Efficient Creation of Stable Mammalian Cell Lines,” Biochem. Biophys. Res. Comm. 229(1):295-298. [cited by applicant]
Helman et al. (Jun. 13, 2016) “Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry,” Cytometry: Part A. 85(2):162-168. [cited by applicant]
Hwang et al. (1998) “Involvement of the 5′ proximal coding sequences of hepatitis C virus with internal initiation of viral translation,” Biochemical and Biophysical Research Communications. 252(2):455-460. [cited by applicant]
International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/US2007/020180, issued Mar. 24, 2009. [cited by applicant]
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2007/020180, mailed May 29, 2008. [cited by applicant]
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2016/055915, mailed Apr. 6, 2017. [cited by applicant]
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2016/055918, mailed Jan. 26, 2017. [cited by applicant]
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2017/055583, mailed Dec. 15, 2017. [cited by applicant]
Jang et al. (1989) “Initiation of Protein Synthesis by Internal Entry of Ribosomes into the 5′ Nontranslated Region of Encephalomyocarditis Virus RNA In Vivo,” J. Virol. 63(4):1651-1660. [cited by applicant]
Jang et al. (1998) “A Segment of the 5′ Nontranslated Region of Encephalomyocarditis Virus RNA Directs Internal Entry of Ribosomes During In Vitro Translation,” J. Virol. 62(8):2636-2643. [cited by applicant]
Jiang et al. (2006) “Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells: A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression,” Biotechnol. Prog. 22:313-318. [cited by applicant]
Kaufman et al. (1990) “Selection and Coamplification of Heterologous Genes in Mammalian Cells”, Methods in Enzymology, 185: 537-566. [cited by applicant]
Keen et al. (1995) “Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line,” Cytotechnology. 17:153-163. [cited by applicant]
Kim et al. (1998) “Characterization of Chimeric Antibody Producing CHO Cells in The Course 0 Dihydrofolate Reductase-Mediated Gene Amplification and Their Stability in The Absence 0 Selective Pressure,” Biotechnol. Bioe… [cited by applicant]
Kim et al. (1998) “Clonal Variability Within Dihydrofolate Reductase-Mediated Gene Amplified Chinese Hamster Ovary Cells: Stability in the Absence of Selective Pressure,” Biotech. Bioeng. 60(6):679-688. [cited by applicant]
Kito et al. (2002) “Construction of Engineered CHO Strains for High-Level Production 0 Recombinant Proteins,” Appl. Microbial. Biotechnol. 60:442-448. [cited by applicant]
Klucher et al. (1997) “A Novel Method to Isolate Cells with Conditional Gene Expression Using Fluorescence Activated Cell Sorting (FACS),” Nucleic Acids Res. 25(23):4858-4860. [cited by applicant]
Knapp et al. (2003) “Detection of 13-Lactamase Reporter Gene Expression by Flow Cytometry,” Cytometry. 51A:68-78. [cited by applicant]
Koller et al. (2001) “A High-Throughput Alphavirus-Based Expression Cloning System for Mammalian Cells,” Nat. Biotechnol. 19:851-855. [cited by applicant]
Kotarsky et al. (2001) “A Chimeric Reporter Gene Allowing for Clone Selection and High-Throughput Screening of Reporter Cell Lines Expressing G-Protein-Coupled Receptors,” Anal. Biochern. 288:209-215. [cited by applicant]
Kozak (1989) “Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems,” Mol. Cell. Biol. 9(11):5073-5080. [cited by applicant]
Kozak (1990) “Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes,” Proc. Natl. Acad. Sci. USA. 87:8301-8305. [cited by applicant]
Kozak (1991) “An analysis of vertebrate mRNA sequences: intimations of translational control,” The Journal of Cell Biology. 115(4):887-903. [cited by applicant]
Liu et al. (2000) “Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels,” Analytical Biochemistry. 280:20-28. [cited by applicant]
Medin et al. (1996) “A Bicistronic Therapeutic Retroviral Vector Enables Sorting of Transduced CD34+ Cells and Corrects the Enzyme Deficiency in Cells from Gaucher Patients,” Blood. 87:1754-1762. [cited by applicant]
Mehdi et al. (1990) “Initiation of Translation at CUG, GUG, and ACG Codones in Mammalian Cells,” Gene. 91:173-178. [cited by applicant]
Meng et al. (2000) “Green Fluorescent Protein as a Second Selectable Marker for Selection of High Producing Clones from Transfected CHO Cells,” Gene. 242:201-207. [cited by applicant]
Mizuguchi et al. (2000) “IRES-Dependent Second Gene Expression is Significantly Lower Thank Cap-Dependent First Gene Expression in a Bicistronic Vector,” Mol. Ther. 1(4):376-382. [cited by applicant]
Mizushima et al. (1990) “pEF-BOS, a powerful mammalian expression vector,” Nucleic Acids Research. 18(17):5322. [cited by applicant]
Mortensen et al. (2009) “Selection of Transfected Mammalian Cells,” Ch. 9. Unit 9.5 In; Curr. Protoc. Mol. Biol. pp. 9.5.1-9.5.13. [cited by applicant]
Moser et al. (2000) “An Update of pTrident Multicistronic Expression Vectors: pTridents Containing Novel Streptogramin-Responsive Promoters,” Biotechnol. Prog. 16:724-735. [cited by applicant]
Mosser et al. (1997) “Use of a Dicistronic Expression Cassette Encoding the Green Fluorescent Protein for the Screening and Selection of Cells Expressing Inducible Gene Products,” BioTechniques. 22:150-161. [cited by applicant]
Pelletier et al. (1988) “Internal Initiation of Translation of Eukaryotic mRNA Directed by a Sequence Derived from Poliovirus RNA,” Nature. 334:320-325. [cited by applicant]
Pestova (1998) “A Prokaryotic-like Mode of Cytoplasmic Eukaryotic Ribosome Binding to the Initiation Codon During Internal Translation Initation of Hepatitis C and Classical Swine Fever Virus? RNAs,” Genes and Developme… [cited by applicant]
Pu et al. (1998) “Rapid Establishment of High-Producing Cell Lines Using Discistronic Vectors With Glutamine Synthetase as The Selection Marker,” Mol. Biotechnol. 10:17-25. [cited by applicant]
Rakestraw et al. (2006) “A Flow Cytometric Assay for Screening Improved Heterologous Protein Secretion in Yeast,” Biotech. Prog. 22(4):1200-1208. [cited by applicant]
Ramesh et al. (1996) “High-Titer Bicistronic Retroviral Vectors Employing Foot-and-Mouth Disease Virus Internal Ribosomes Entry Site,” Nucleic Acids Research. 24(14):2697-2700. [cited by applicant]
Rees et al. (1996) “Bicistronic Vector for the Creation of Stable Mammalian Cell Lines that Predisposes all Antibiotic-Resistant Cells to Express Recombinant Protein,” Biotechniques. 20:102-110. [cited by applicant]
Sato et al. (Dec. 5, 2014) “A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells,” Biotechnol. J. 10(1… [cited by applicant]
Sato et al. (Mar. 2013) “Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression,” Biology. 2(1):341-355. [cited by applicant]
Sautter et al. (2005) “Selection of High-Producing CHO Cells Using NPT Selection marker with Reduced Enzyme Activity,” Biotech. Bioeng. 89(5):530-538. [cited by applicant]
Scharfenberg et al. (1995) “A Reliable Strategy for The Achievement of Cell Lines Growing in Protein-Free Medium,” In; Animal Cell Technology: Developments Towards the 21st Century. Eds.: Beuvery et al. pp. 619-623. [cited by applicant]
Schlatter et al. (2001) “Novel surface tagging technology for selection of complex proliferation-controlled mammalian cell phenotypes,” Biotechnology and Bioengineering. 75(5):597-606. [cited by applicant]
Schlatter et al. (2005) “On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells,” Biotech Prog. 21:122-133. [cited by applicant]
Schubert et al. (1999) “Regulation of Virus Release by the Macrophage-Tropic Human Immunodeficiency Virus Type 1 AD8 Isolate is Redundant and can be Controlled by either Vpu or Env,” Journal of Virology. 73(2):887-896. [cited by applicant]
Sen et al. (1990) “Flow Cytometric Study of Hybridoma Cell Culture: Correlation Between Cell Surface Fluorescence and IgG Production Rate,” Enzyme Microb. Technol. 12:571-576. [cited by applicant]
Soriano et al. (2002) “Optimization of Recombinant Protein Expression Level in [cited by applicant]
Tsai et al. (2002) “Evidence for translational regulation of the imprinted Snurf-Snrpn locus in mice,” Human Molecular Genetics. 11(14):1659-1668. [cited by applicant]
Urlaub et al. (1980) “Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity,” Proc. Natl. Acad. Sci. USA. 77(7):4216-4220. [cited by applicant]
Yoshikawa et al. (2001) “Flow Cytometry: An Improved Method for the Selection of Highly Productive Gene-Amplified CHO Cells Using Flow Cytometry,” Biotechnol. Bioeng. 74(5):435-442. [cited by applicant]
Yuk et al. (2002) “A GFP-Based Screen for Growth-Arrested, Recombinant Protein-Producing Cells,” Biotechnol. Bioeng. 79(1):74-82. [cited by applicant]
Zeyda et al. (1999) “Optimization of Sorting Conditions for the Selection of Stable, High-Producing Mammalian Cell Lines,” Biotechnol. Prog. 15:953-957. [cited by applicant]
Zlokarnik et al. (1998) “Quantitation of Transcription and Clonal Selection of Single Living Cells With 13-Lactamase as Reporter,” Science. 279:84-88. [cited by applicant]
U.S. Appl. No. 12/441,806 2009/0239235, filed Mar. 18, 2009 Sep. 24, 2009, Christine DeMaria, Facs- and Reporter Protein-Based System for High Throughput Development of Therapeutic Proteins. [cited by applicant]
U.S. Appl. No. 15/288,036 2017/0227441 U.S. Pat. No. 11,635,363, filed Oct. 7, 2016 Aug. 10, 2017 Apr. 25, 2023, Victor R. Cairns, Flare (Flow Cytometry Attenuated Reporter Expression) Technology for Rapid Bulk Sorting. [cited by applicant]
2023/0304913, Mar. 10, 2023 Sep. 28, 2023, Victor R. Cairns, Flare (Flow Cytometry Attenuated Reporter Expression) Technology for Rapid Bulk Sorting. [cited by applicant]
U.S. Appl. No. 15/288,050 2017/0121734 U.S. Pat. No. 10,317,329, filed Oct. 7, 2016 May 4, 2017 Jun. 11, 2019, Victor R. Cairns, Flare (Flow Cytometry Attenuted Reporter Expression) Technology for Rapid Bulk Sorting. [cited by applicant]
U.S. Appl. No. 16/392,174 2020/0018682, filed Apr. 23, 2019 Jan. 16, 2020, Victor R. Cairns, Early Post-Transfection Isolation of Cells (Epic) for Biologics Production. [cited by applicant]
U.S. Appl. No. 15/727,272 2018/0119192, filed Oct. 6, 2017 May 3, 2018, Victor R. Cairns, Early-Post Transfection Isolation of Cells (Epic) for Biologics Production. [cited by applicant]
U.S. Appl. No. 16/683,837 2020/0299743 U.S. Pat. No. 11,685,943, filed Nov. 14, 2019 Sep. 24, 2020 Jun. 27, 2023, Victor R. Cairns, Early Port-Transfection Isolation of Cells (Epic) for Biologics Production. [cited by applicant]
U.S. Appl. No. 18/315,332 2024/0026410, filed May 10, 2023 Jan. 25, 2024, Victor R. Cairns, Early Post-Transfection Isolation of Cells (Epic) for Biologics Production. [cited by applicant]