CHO cells; next-generation sequencing; sequence variants; single-molecule real-time sequencing; Biological Products; DNA, Recombinant; Recombinant Proteins; Green Fluorescent Proteins; Animals; Biological Products/metabolism; CHO Cells; Computational Biology/methods; Cricetulus; DNA, Recombinant/genetics; Green Fluorescent Proteins/genetics; Plasmids; Recombinant Proteins/genetics; Sequence Analysis, DNA/methods; Technology, Pharmaceutical/methods; Mutation; CHO cell; Highly sensitive detections; Mammalian cell culture; Quantitative assessments; Real time; Recombinant gene expressions; Computational Biology; Sequence Analysis, DNA; Technology, Pharmaceutical; Biotechnology; Bioengineering; Applied Microbiology and Biotechnology
Abstract :
[en] High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process.
Disciplines :
Biotechnology
Author, co-author :
Cartwright, Joseph F ; Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK
Aggarwal, R. S. (2014). What's fueling the biotech engine-2012 to 2013. Nature Biotechnology, 32, 32–39.
Ahmadi, M., Damavandi, N., Akbari, M. R. E., & Davami, F. (2016). Utilization of Site-Specific Recombination in Biopharmaceutical Production. Iranian Biomedical Journal, 20, 68–76.
Arad, U. (1998). Modified Hirt procedure for rapid purification of extrachromosomal DNA from mammalian cells. BioTechniques, 24, 760-+.
Brodin, J., Mild, M., Hedskog, C., Sherwood, E., Leitner, T., Andersson, B., & Albert, J. (2013). PCR-induced transitions are the major source of error in cleaned ultra-deep pyrosequencing data. PLoS ONE, 8(7), e70388.
Bull, R. A., Eden, J. S., Luciani, F., Mcelroy, K., Rawlinson, W. D., & White, P. A. (2012). Contribution of intra- and interhost dynamics to norovirus evolution. Journal of Virology, 86, 3219–3229.
Chaisson, M. J., & Tesler, G. (2012). Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): Application and theory. BMC Bioinformatics, 13(1), 238.
Daum, L. T., Rodriguez, J. D., Worthy, S. A., Ismail, N. A., Omar, S. V., Dreyer, A. W., … Fischer, G. W. (2012). Next-generation ion torrent sequencing of drug resistance mutations in mycobacterium tuberculosis strains. Journal of Clinical Microbiology, 50, 3831–3837.
Deaven, L. L., & Petersen, D. F. (1973). Chromosomes of CHO, an aneuploid Chinese-hamster cell line − g-band, c-band, and autoradiographic analyses. Chromosoma, 41, 129–144.
Dejong, P. J., Grosovsky, A. J., & Glickman, B. W. (1988). Spectrum of spontaneous mutation at the APRT locus of Chinese-hamster ovary cells − an analysis at the DNA-sequence level. Proceedings of the National Academy of Sciences of the United States of America, 85, 3499–3503.
Derouazi, M., Martinet, D., Besuchet Schmutz, N., Flaction, R., Wicht, M., Bertschinger, M., … Wurm, F. M. (2006). Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. Biochemical and Biophysical Research Communications, 340, 1069–1077.
Dinnis, D. M., & James, D. C. (2005). Engineering mammalian cell factories for improved recombinant monoclonal antibody production: Lessons from nature? Biotechnology and Bioengineering, 91, 180–189.
Ewing, B., & Green, P. (1998). Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research, 8, 186–194.
Ewing, B., Hillier, L., Wendl, M. C., & Green, P. (1998). Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research, 8, 175–185.
Feichtinger, J., Hernandez, I., Fischer, C., Hanscho, M., Auer, N., Hackl, M., … Borth, N. (2016). Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time. Biotechnology and Bioengineering, 113, 2241–2253.
Flaherty, P., Natsoulis, G., Muralidharan, O., Winters, M., Buenrostro, J., Bell, J., … Ji, H. P. (2012). Ultrasensitive detection of rare mutations using next-generation targeted resequencing. Nucleic Acids Research, 40(1), e2–e2.
Frye, C., Deshpande, R., Estes, S., Francissen, K., Joly, J., Lubiniecki, A., … Anderson, K. (2016). Industry view on the relative importance of “clonality” of biopharmaceutical-producing cell lines. Biologicals, 44, 117–122.
Gates, K. S. (2009). An overview of chemical processes that damage cellular DNA: Spontaneous hydrolysis, alkylation, and reactions with radicals. Chemical Research in Toxicology, 22, 1747–1760.
Gojobori, T., Li, W. H., & Graur, D. (1982). Patterns of nucleotide substitution in pseudogenes and functional genes. Journal of Molecular Evolution, 18, 360–369.
Goulian, M., Bleile, B., & Tseng, B. Y. (1980). Methotrexate-induced misincorporation of uracil into DNA. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences, 77, 1956–1960.
Greaves, M., & Maley, C. C. (2012). Clonal evolution in cancer. Nature, 481, 306–313.
Guo, D. L., Gao, A., Michels, D. A., Feeney, L., Eng, M., Chan, B., … Shen, A. (2010). Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells. Biotechnology and Bioengineering, 107, 163–171.
Guye, P., Li, Y. Q., Wroblewska, L., Duportet, X., & Weiss, R. (2013). Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Research, 41(16), e156–e156.
Harris, R. J., Murnane, A. A., Utter, S. L., Wagner, K. L., Cox, E. T., Polastri, G. D., … Sliwkowski, M. B. (1993). Assessing genetic-heterogeneity in production cell-lines − detection by peptide-mapping of a low-level TYRr to GLN sequence variant in a recombinant antibody. Bio-Technology, 11, 1293–1297.
Hauser, J., Levine, A. S., & Dixon, K. (1987). Unique pattern of point mutations arising after gene transfer into mammalian cells. The EMBO Journal, 6, 63–67.
Heller-Harrison, R., Crowe, K., Cooley, C., Hone, M., Mccarthy, K., & Leonard, M. (2009). Managing cell line instability and its impact during cell line development. Biopharm International, 22, 16–27.
Hodgkinson, A., & Eyre-Walker, A. (2011). Variation in the mutation rate across mammalian genomes. Nature Reviews Genetics, 12, 756–766.
Huang, D. W., Raley, C., Jiang, M. K., Zheng, X., Liang, D., Rehman, M. T., … Dewar, R. L. (2016). Towards better precision medicine: Pacbio single-molecule long reads resolve the interpretation of HIV drug resistant mutation profiles at explicit quasispecies (Haplotype) level. Journal of Data Mining in Genomics & Proteomics, 7, 182.
Huang, Y. M., Hu, W. W., Rustandi, E., Chang, K., Yusuf-Makagiansar, H., & Ryll, T. (2010). Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Biotechnology Progress, 26, 1400–1410.
Jayapal, K. R., Wlaschin, K. F., Hu, W-S., & Yap, M. G. S. (2007). Recombinant protein therapeutics from CHO cells − 20 years and counting. Cell Engineering Progress, 103, 40–47.
Kildegaard, H. F., Baycin-Hizal, D., Lewis, N. E., & Betenbaugh, M. J. (2013). The emerging CHO systems biology era: Harnessing the 'omics revolution for biotechnology. Current Opinion in Biotechnology, 24, 1102–1107.
Kim, J. Y., Kim, Y. G., & Lee, G. M. (2012). CHO cells in biotechnology for production of recombinant proteins: Current state and further potential. Applied Microbiology and Biotechnology, 93, 917–930.
Kim, M., O'callaghan, P. M., Droms, K. A., & James, D. C. (2011). A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnology and Bioengineering, 108, 2434–2446.
Kimura, M. (1955). Solution of a process of random genetic drift with a continuous model. Proceedings of the National Academy of Sciences of the United States of America, 41, 144–150.
Kimura, M. (1979). Model of effectively neutral mutations in which selective constraint is incorporated. Proceedings of the National Academy of Sciences of the United States of America, 76, 3440–3444.
Lebkowski, J. S., Dubridge, R. B., Antell, E. A., Greisen, K. S., & Calos, M. P. (1984). Transfected DNA is mutated in monkey, mouse, and human-cells. Molecular and Cellular Biology, 4, 1951–1960.
Lee, H., Popodi, E., Tang, H., & Foster, P. L. (2012). Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. Proceedings of the National Academy of Sciences of the United States of America, 109, E2774.
Lee, J. S., Kallehauge, T. B., Pedersen, L. E., & Kildegaard, H. F. (2015). Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports, 5, 8572.
Lewis, N. E., Liu, X., Li, Y., Nagarajan, H., Yerganian, G., O'brien, E., … Palsson, B. O. (2013). Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nature Biotechnology, 31, 759.
Li, F., Vijayasankaran, N., Shen, A. Y., Kiss, R., & Amanullah, A. (2010). Cell culture processes for monoclonal antibody production. mAbs, 2, 466–479.
Loeb, L. A. (2011). Human cancers express mutator phenotypes: Origin, consequences and targeting. Nature Reviews Cancer, 11, 450–457.
Martin, S. A., Mccarthy, A., Barber, L. J., Burgess, D. J., Parry, S., Lord, C. J., & Ashworth, A. (2009). Methotrexate induces oxidative DNA damage and is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2. Embo Molecular Medicine, 1, 323–337.
Matsuda, T., Matsuda, S., & Yamada, M. (2015). Mutation assay using single-molecule real-time (SMRT(TM)) sequencing technology. Genes and Environment: The Official Journal of the Japanese Environmental Mutagen Society, 37, 15.
McCarthy, A. (2010). Third generation DNA sequencing: Pacific biosciences' single molecule real time technology. Chemistry & Biology, 17, 675–676.
McCulloch, S. D., & Kunkel, T. A. (2008). The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. Cell Research, 18, 148–161.
McElroy, K., Thomas, T., & Luciani, F. (2014). Deep sequencing of evolving pathogen populations: Applications, errors, and bioinformatic solutions. Microbial Informatics and Experimentation, 4, 1–1.
Pilbrough, W., Munro, T. P., & Gray, P. (2009). Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS ONE, 4(12), e8432.
Prentice, H. L., Ehrenfels, B. N., & Sisk, W. P. (2007). Improving performance of mammalian cells in fed-batch processes through “bioreactor evolution“. Biotechnology Progress, 23, 458–464.
Pybus, L. P., Dean, G., West, N. R., Smith, A., Daramola, O., Field, R., … James, D. C. (2014). Model-directed engineering of “difficult-to-express“ monoclonal antibody production by Chinese hamster ovary cells. Biotechnology and Bioengineering, 111, 372–385.
Ren, D., Zhang, J., Pritchett, R., Liu, H., Kyauk, J., Luo, J., & Amanullah, A. (2011). Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 879, 2877–2884.
Roberts, R. J., Carneiro, M. O., & Schatz, M. C. (2013). The advantages of SMRT sequencing. Genome Biology, 14(6), 405.
Shah, S. P., Morin, R. D., Khattra, J., Prentice, L., Pugh, T., Burleigh, A., … Aparicio, S. (2009). Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature, 461, 809–U67.
Sinacore, M. S., Drapeau, D., & Adamson, S. R. (2000). Adaptation of mammalian cells to growth in serum-free media. Molecular Biotechnology, 15, 249–257.
Talundzic, E., Plucinski, M. M., Biliya, S., Silva-Flannery, L. M., Arguin, P. M., Halsey, E. S., … Udhayakumar, V. (2016). Advanced molecular detection of malarone resistance. Antimicrobial Agents and Chemotherapy, 60, 3821–3823.
Tomlinson, I. P. M., Novelli, M. R., & Bodmer, W. F. (1996). The mutation rate and cancer. Proceedings of the National Academy of Sciences of the United States of America, 93, 14800–14803.
Travers, K. J., Chin, C. S., Rank, D. R., Eid, J. S., & Turner, S. W. (2010). A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Research, 38(15), e159–e159.
van Berkel, P. H. C., Gerritsen, J., Perdok, G., Valbjorn, J., Vink, T., Van DE Winkel, J. G. J., & Parren, P. W. H. I. (2009). N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG. Biotechnology Progress, 25, 244–251.
Vazquez-Rey, M., & Lang, D. A. (2011). Aggregates in monoclonal antibody manufacturing processes. Biotechnology and Bioengineering, 108, 1494–1508.
Walsh, G. (2014). Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32, 992–1000.
Wen, D., Vecchi, M. M., Gu, S., Su, L., Dolnikova, J., Huang, Y.-M., … Meier, W. (2009). Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells. Journal of Biological Chemistry, 284, 32686–32694.
Westerhoff, H. V., & Palsson, B. O. (2004). The evolution of molecular biology into systems biology. Nature Biotechnology, 22, 1249–1252.
Wright, C., Groot, J., Swahn, S., Mclaughlin, H., Liu, M., Xu, C., … Estes, S. (2016). Genetic mutation analysis at early stages of cell line development using next generation sequencing. Biotechnology Progress, 32(3), 813–817.
Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology, 22, 1393–1398.
Wurm, F. M., & Hacker, D. (2011). First CHO genome. Nature Biotechnology, 29, 718–720.
Yates, L. R., & Campbell, P. J. (2012). Evolution of the cancer genome. Nature Reviews Genetics, 13, 795–806.
Yu, X. C., Borisov, O. V., Alvarez, M., Michels, D. A., Wang, Y. J., & Ling, V. (2009). Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry. Analytical Chemistry, 81, 9282–9290.
Zhang, J. (2010). Mammalian cell culture for biopharmaceutical production. Manual of Industrial Microbiology and Biotechnology, Third Edition, 157–178.
Zhang, S., Bartkowiak, L., Nabiswa, B., Mishra, P., Fann, J., Ouellette, D., … Liu, J. (2015). Identifying low-level sequence variants via next generation sequencing to aid stable CHO cell line screening. Biotechnology Progress, 31, 1077–1085.
Zhu, J. (2012). Mammalian cell protein expression for biopharmaceutical production. Biotechnology Advances, 30, 1158–1170.
