Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing.

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines. We further demonstrate a correlation between specific productivity (qP) and intracellular heavy chain (HC) content with final productivity. The unique combination of screening using ClonePix FL and the flow cytometry approaches facilitated more efficient isolation of clonal cell lines with high productivity within a 15week timeline and which can be applied across NS0 and CHO host platforms. Furthermore, in this study we describe the critical parameters for the ClonePix FL colony based selection and the associated calculations to provide an assessment of the probability of monoclonality of the resulting cell lines.

[1]  M. Tellers,et al.  Development of transfection and high-producer screening protocols for the CHOK1SV cell system , 2006, Molecular biotechnology.

[2]  Takeshi Omasa,et al.  Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system. , 2015, Journal of bioscience and bioengineering.

[3]  W. Bonner,et al.  Cell Sorting: Automated Separation of Mammalian Cells as a Function of Intracellular Fluorescence , 1969, Science.

[4]  M. Brigido,et al.  Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells , 2010, Molecular biotechnology.

[5]  R. Pérez,et al.  Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells , 2011, Cytotechnology.

[6]  C. Demaria,et al.  Accelerated Clone Selection for Recombinant CHO Cells Using a FACS‐Based High‐Throughput Screen , 2007, Biotechnology progress.

[7]  F. Wurm.  Production of recombinant protein therapeutics in cultivated mammalian cells , 2004, Nature Biotechnology.

[8]  Benjamin S. Hughes,et al.  High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system. , 2014, New biotechnology.

[9]  G. Fassina,et al.  Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains. , 1996, The EMBO journal.

[10]  Kristi Daris,et al.  Cell line profiling to improve monoclonal antibody production , 2014, Biotechnology and bioengineering.

[11]  I. Wilson,et al.  A model for bacterial conjugal gene transfer on solid surfaces. , 2003, FEMS microbiology ecology.

[12]  A. Dickson,et al.  Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells , 2004, Biotechnology and bioengineering.

[13]  H. Katinger,et al.  Changes during subclone development and ageing of human antibody-producing recombinant CHO cells. , 1999, Journal of biotechnology.

[14]  P. Jhurani,et al.  Nucleic acid capture assay, a new method for direct quantitation of nucleic acids. , 2003, Nucleic acids research.

[15]  J. Bailey,et al.  Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells. , 1989, Journal of immunological methods.

[16]  A. Garnier,et al.  Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins , 2009, BMC biotechnology.

[17]  Joni Tsukuda,et al.  A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies , 2009, Biotechnology and bioengineering.

[18]  D. King,et al.  High-Level Expression of a Recombinant Antibody from Myeloma Cells Using a Glutamine Synthetase Gene as an Amplifiable Selectable Marker , 1992, Bio/Technology.

[19]  P Koltay,et al.  Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation. , 2015, Biosensors & bioelectronics.

[20]  J. Maizel,et al.  Synthesis of Excess Light Chains of Gamma Globulin by Rabbit Lymph Node Cells , 1966, Nature.

[21]  R. Schimke,et al.  Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line , 1981, Molecular and cellular biology.

[22]  G. M. Lee,et al.  Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. , 1998, Biotechnology and bioengineering.

[23]  A. Racher,et al.  On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells , 2008, Biotechnology progress.

[24]  C. Frye,et al.  Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines. , 2016, Biologicals : journal of the International Association of Biological Standardization.

[25]  M. Betenbaugh,et al.  Early prediction of instability of chinese hamster ovary cell lines expressing recombinant antibodies and antibody‐fusion proteins , 2012, Biotechnology and bioengineering.

[26]  G. Chiang,et al.  A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. , 2003, Journal of immunological methods.

[27]  S. Sharfstein,et al.  Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells: A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression , 2006, Biotechnology progress.

[28]  J. H. Kim,et al.  Flow cytometric analysis of antibody producing cells using double immunofluorescent staining , 1996 .

[29]  Suba Dharshanan,et al.  Screening and subcloning of high producer transfectomas using semisolid media and automated colony picker. , 2014, Methods in molecular biology.

[30]  H. Coller,et al.  Statistical analysis of repetitive subcloning by the limiting dilution technique with a view toward ensuring hybridoma monoclonality. , 1983, Hybridoma.

[31]  Guillermo Miró-Quesada,et al.  Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality , 2018, Biotechnology progress.

[32]  Johannes Buchner,et al.  How antibodies fold. , 2010, Trends in biochemical sciences.

[33]  David P. Kreil,et al.  Transcriptomic changes in CHO cells after adaptation to suspension growth in protein-free medium analysed by a species-specific microarray. , 2017, Journal of biotechnology.

[34]  M. Fussenegger,et al.  Genetic optimization of recombinant glycoprotein production by mammalian cells. , 1999, Trends in biotechnology.

[35]  N. Mermod,et al.  IL‐17F co‐ ;expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering , 2013, Biotechnology and bioengineering.