Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally‐derived or not

Abstract

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:559-569, 2018.

High‐throughput screening of antibody‐expressing CHO clones using an automated shaken deep‐well system

“…Haines et al presented on an automated liquid handling system to maintain cell lines during evaluation but did not integrate titer and cell density determination into the platform. Automation is also being introduced earlier in the cell line development process for imaging single cells deposited into wells of multi‐well plates to satisfy regulatory requirements to demonstrate clonal‐derivation of cell lines . Furthermore, miniaturization of the cell line screening processes with advances in microfluidic and nanofluidic instruments is underway to allow evaluation of large numbers of clones …”

“…Successive screening steps narrow the initial pool of cell lines to a small number for evaluation in bioreactors to choose the final clone for production of clinical and commercial therapeutic protein product . Production cell lines are clonally‐derived from single cells to meet regulatory expectations and require detailed characterization of the cloning process to determine probability of monoclonality …”

A novel mammalian cell line development platform utilizing nanofluidics and optoelectro positioning technology

“…First, single‐cell isolation is inefficient. Single cells are deposited in individual wells of a cell culture plate by limiting dilution, single cell printing, or fluorescence‐activated cell sorting (FACS) . Each of these methods have degrees of error in isolating individual cells and clonality must be verified by manual imaging of each individual well, followed by visual confirmation of the presence of a single cell in the well .…”

“…OEP can manipulate hundreds of cells in parallel on OptoSelect™ Chips, allowing multiplexed depositing of single cells into an array of individual pens with nanoliter volumes (nanopen). The entire chip can be imaged in 8 min, and coupled with artificial intelligence‐based cell counting, allows for efficient monitoring of clonality and growth of all clones on the chip . Additionally, bead and diffusion‐based fluorescent assays can be adapted for scoring secreted antibody on chip.…”

Methods for Estimating the Probability of Clonality in Cell Line Development

“…Regulatory agencies generally expect that mammalian cell lines expressing the desired therapeutic biologic that are used in commercial manufacturing be derived from a single progenitor cell and considered “monoclonal.” The ICH Q5D guideline specifies the requirement for clonality, as “… the cell substrate … has been cloned from a single cell progenitor.” And the EMA/CHMP guidance requests that “… the cell substrate to be used … should be a stable and continuous monoclonal cell line …” One interpretation for this requirement is that a clonally derived cell line is likely to be more homogenous and could potentially allow for more consistent cell culture performance and product quality . However, this assumption has been recently challenged as growing evidence has shown that a cell bank derived from a single progenitor cell doesn't guarantee cell homogeneity and stability .…”