Note split linear-log axes. is of considerable interest to industry yet MPI-0479605 the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection. Introduction Protein biologics are an important and growing segment of the drug industry with over US$80 billion in sales worldwide. Many protein biologics, including monoclonal antibodies, are large, structurally-complex glycoproteins requiring functional human-like post-translational modifications for their activity [1]. Cultured mammalian cells, and particularly Chinese hamster ovary (CHO) cells [2], are generally employed as production hosts because simpler prokaryotic and eukaryotic expression systems lack suitable native glycosylation machinery and may not fold and secrete these biomolecules efficiently [3]. Yet despite their widespread use and commercial significance, two major issues remain unresolved in establishing productive mammalian cell lines, namely clonal heterogeneity [4] and expression instability [5]. Large-scale production of recombinant proteins relies on stable integration of expression vectors into the host genome [6]. Ordinarily this involves non-targeted DNA delivery and chemical selection to integrate and amplify transgene sequences encoding the product [7]. The resulting transfectants differ markedly in expression due to an inherent lack of control over gene dosage and chromosomal context of integrating copies [8]C[10]. Random integration and amplification may also disrupt or dysregulate endogenous genes MPI-0479605 [11]C[13] creating the potential for variation in other cell traits [14], [15]. Accordingly, production cell lines are cloned, or derived from a single cell, in order to minimize heterogeneity (International Conference on Harmonisation (ICH), Guideline Q5D, 1997). Upstream MPI-0479605 of the cloning step, however, the marked diversity amongst transfectants makes the process of clone isolation a considerable challenge. High producers are rare and those also satisfying product quality and other selection criteria, such as rapid growth, are rarer MPI-0479605 still [6]. Extensive empirical screening of large numbers of candidate clones is therefore required, which is resource intensive and frequently rate limiting in early development. Protein expression stability also tends to be problematic. Most clones suffer a decline in productivity during the extended culture periods required to reach manufacturing scale, yet this is unpredictable and varies from clone to clone. Efforts to define the molecular determinants of stability [16] have so far achieved only limited success and stability is still routinely assessed by directly monitoring each clone over several months of growth. Prior examination of these issues has focused chiefly on differences clones isolated from mixed populations, such as those arising from transfection or gene amplification [4], [17]C[24]. We take an alternate approach, exploring the degree of variation a clone, using single cell analysis facilitated by IRES-driven coexpression of intracellular fluorescent markers. Clones are normally assumed to be homogeneous but emerging fundamental research in bacteria MYO7A [25]C[28], yeast [29]C[34], and more recently mammalian cells [35]C[39], has revealed that gene expression can vary significantly between genetically-identical cells,.
