Analysis of heterogeneity and instability of stable mAb-expressing CHO cells

Du, Zhimei; Mujacic, Mirna; Le, Kim; Caspary, Guy; Nunn, Heather; Heath, Carole A.; Reddy, Pranhitha

doi:10.1007/s12257-012-0577-1

Cited by 17 publications

(11 citation statements)

References 19 publications

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“…Indeed, the measured characteristics, such as titer, VCD, copy number and HMW of the originating MCB are around the average of the subclones for each measured parameter (Figures and ). To further confound these observations, subcloning involves exposure to shear forces within the FACS instrument, followed by outgrowth in media to support low density growth which can change selection pressures and induce population changes . Therefore, the act of subcloning itself could amplify the differences observed in the population, resulting in subclone profiles more diverse than existed in the original clonal cell line.…”

Section: Discussionmentioning

confidence: 99%

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Tharmalingam

Barkhordarian

Tejeda

et al. 2018

Biotechnology Progress

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Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long‐term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single‐cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub‐cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613–623, 2018

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Section: Discussionmentioning

confidence: 99%

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Tharmalingam

Barkhordarian

Tejeda

et al. 2018

Biotechnology Progress

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“…Although this method has the significant advantages of higher product yields compared to the transient format, and faster speed over standard cell line construction, such pools are subject to declining productivity with increasing number of population doublings as non-productive cells in the pool overgrow the original population . A study by Du et al (2013) analysed the heterogeneity present within MTX-amplified DHFR-CHO transfectant pools and the effect of serial sub-culture by assessment of the Mab expression at the cell surface using flow cytometry. Most of the pools assessed during the study showed a normal (Gaussian) distribution in terms of cell surface Mab content (a proxy measurement for overall expression), characterised by a single peak encompassing approximately an order of magnitude difference in Mab expression.…”

Section: Impact Of Long-term Sub-culture On Cell Populationsmentioning

confidence: 99%

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

O'Callaghan¹,

Racher

2014

Cell Engineering

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Considerable effort is expended during the mammalian cell line construction process to find a stably-transfected clone capable of supporting the largescale manufacture of a recombinant therapeutic protein. Such a clone must synthesise a sufficient volumetric concentration of the product with the correct biochemical characteristics (glycosylation, structural integrity, etc.). Furthermore, this performance must be maintained over the extended time period required to support a manufacturing campaign in 20,000 L bioreactors. However, a significant proportion of recombinant clonal cell lines show production instability over longterm sub-culture, where volumetric product yield and/or product quality are not maintained. This instability can potentially extend product development timelines and can affect the ability of a manufacturing process to meet market demand. In the worse-case scenario it can also jeopardise patient safety if product quality is impaired. In order to prevent this, industrial cell line construction processes include long-term stability studies where several candidate lead clones are serially sub-cultured and monitored for signs of instability before selecting the final production cell line. The roots of production instability are varied, but the epigenetic silencing of transgenes at sites of host cell chromosome integration, and the direct mutation or loss of transgenes are prominent molecular causes. Considerable research has been conducted by both industry and academic groups into the molecular mechanisms underpinning instability, with an emphasis on uncovering early predictive markers of incipient instability as well as preventing its occurrence. In this article we present a detailed overview of the industrial experience of production instability and its impact on the manufacturing of recombinant therapeutics. We also discuss our current understanding of the molecular causes of cell line instability and how this has been used to mitigate the impact of this phenomenon through novel vector redesigns and cell line screening.

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“…The requirement for clonality is based on the assumption that subclones are phenotypically and genetically homogenous and consist of uniform and identical cells. Nevertheless, clones may experience phenotypic drifts, diverging growth and production properties, and with each division re‐acquire heterogeneity . It was also shown that resubcloning a confirmed subclone will again lead to subclones with diverse properties …”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, clones may experience phenotypic drifts, diverging growth and production properties, and with each division re-acquire heterogeneity. [9][10][11] It was also shown that resubcloning a confirmed subclone will again lead to subclones with diverse properties. [12] The aberrant karyotypes as well as chromosomal rearrangements can be analyzed by cytogenetic methods.…”

Section: Introductionmentioning

confidence: 99%

Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning

Vcelar

Melcher

Auer

et al. 2018

Biotechnology Journal

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Chinese hamster ovary (CHO) cells are the number one production system for therapeutic proteins. A pre-requirement for their use in industrial production of biopharmaceuticals is to be clonal, thus originating from a single cell in order to be phenotypically and genomically identical. In the present study it was evaluated whether standard procedures, such as the generation of a recombinant cell line in combination with selection for a specific and stable phenotype (expression of the recombinant product) or subcloning have any impact on karyotype stability or homogeneity in CHO cells. Analyses used were the distribution of chromosome counts per cell as well as chromosome painting to identify specific karyotype patterns within a population. Results indicate that subclones both of the host and the recombinant cell line are of comparable heterogeneity and (in)stability as the original pool. In contrast, the rigorous selection for a stably expressing phenotype generated cell lines with fewer variation and more stable karyotypes, both at the level of the sorted pool and derivative subclones. We conclude that the process of subcloning itself does not contribute to an improved karyotypic homogeneity of a population, while the selection for a specific cell property inherently can provide evolutionary pressure that may lead to improved chromosomal stability as well as to a more homogenous population.

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Analysis of heterogeneity and instability of stable mAb-expressing CHO cells

Cited by 17 publications

References 19 publications

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning

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