Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity

Abstract

Routine CHO cell line development practices involve a lengthy process of iteratively screening clonally derived cell lines to identify a single line suitable for IND filing and clinical manufacture. Paramount in this process is development of a stable production cell line having consistent growth, productivity and product quality for the entire generational length of the manufacturing process. Scale-down stability models used to screen clones for consistency are time consuming and often a rate-limiting step in clone selection. To investigate CHEF1 production stability in CHO cells we analyzed genotypic and phenotypic attributes of monoclonal primary clones and their respective subclones over time in standard antibody production models. The main finding of this work indicates that monoclonal cell lines derived from single cell progenitors grow into populations of cells with varied phenotypic heterogeneity, as revealed in their subclones, from either stable or unstable cell lines. Investigation of the subclones demonstrates that clonally derived cell lines grow out into populations with variable phenotypes and genotypes, even if the primary clone shows consistency in both over many generations in a stability study. Phenotypic and genotypic heterogeneity mostly did not correlate, but growth and productivity appear driven in part by cytosine methylation heterogeneity in both primary and secondary clones. This work presents evidence that epigenetic analysis may be useful for early detection of stability traits, but emphasizes the continued importance of rigorous cell line stability screening to identify primary clones that have consistent phenotypic characteristics, especially growth and productivity, throughout the in vitro lifecycle of the cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:635-649, 2018.

Quantification of the dynamics of population heterogeneities in CHO cultures with stably integrated fluorescent markers

“…However, population heterogeneities can significantly affect the bioprocess performance, e.g., with differences in the intraclonal protein expression or metabolically different cell populations [4][5][6][7][8]. It was shown that even in monoclonal CHO cell lines ( [9], recently discussed in [10]), population heterogeneities with different phenotypes can spontaneously evolve [4,[11][12][13], and that cell-cycle-dependent population dynamics with variable productivities and metabolic regulations can be present in cell culture processes [14][15][16][17]. The formation and dynamics of physically-derived process heterogeneities are studied intensively in bioprocesses, e.g., evaluating spatial gradients of gas bubbles [18], concentration gradients of substrates [19] and oxygen [20], and pH gradients [21].…”

A New Cell Model for Investigating Prion Strain Selection and Adaptation

“…Single cell cloning is an excellent way to limit genetic variation, while harnessing phenotypic heterogeneity to establish favorable cell models. Phenotypic heterogeneity of clonally derived cells is often associated with epigenetic variation [41] and triggered by stochastic gene activation events [42], which is an important contributor to reprogramming transitions [43,44]. Systematic studies into stochastic gene expression events have been facilitated by single cell transcriptomics (for recent reviews see [44,45,46]).…”

Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data

“…Many of the techniques and examples we present may pose a challenge to companies, as new questions arise concerning how the findings could affect a product or program. Given the well-documented plasticity of CHO cells, detailed genetic examination of legacy cell lines runs the risk of generating data that are not easily explained, or even dispute clonality of a cell line that is, in fact, clonal in origin (14)(15)(16)(17). This additional information may contradict the breadth of knowledge that supports a cell line's commercial use, such as extensive characterization, or phenotypic and genotypic stability evaluations.…”

“…Also, the potential for genetic rearrangements applies and should be considered when designing experiments and interpreting data. However, TLA can be very useful to generate markers for clonality, such as the unique transgene to CHO genomic DNA (TG-CHO) fusions that arise at integration (16). These TG-CHO fusion junctions can be detected by simpler PCR-based methods to follow clonal lineages, as detailed in Section 3.5.…”