Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging

Abstract

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low‐density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 31:1172–1178, 2015

Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

“…Cell Metric CLD is an automated, high resolution, high‐throughput system providing fast whole well imaging capability. In conjunction with other similar optical systems, it has been widely used for monoclonality evidence generation (Figure d), which is required for the cell lines intended for the recombinant protein production in the GMP environment . Here, we developed a cell counting application based on Cell Metric CLD imaging analysis software for determining total cell count and growth patterns of individual clones.…”

“…The clones were evaluated at the 1 to ~2,000 cell stage during the initial 14‐day colony expansion window. The following clone characteristics were established: (a) single‐cell FACS sorting and automated 96‐well plate imaging were used as orthogonal high‐throughput approaches to ensure the clone monoclonality, with probabilities exceeding 99%; (b) total cell counts were estimated between Day 0 (a single cell deposition) and Day 14 providing growth rates of the clones; (c) VP and qP were estimated for each clone; (d) the epitope profiling of the secreted antigen was performed using multiplex Luminex platform. The quantitative cell growth, productivity, and product quality data obtained in 96‐well plate culture format allowed us to rapidly narrow down the lead candidate clone set and proceed directly to the clonal expansion and fed‐batch evaluation.…”

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity

“…Bulk cell sorting and single-cell deposition cloning was performed using a BD Influx cell sorter (BD Biosciences) based on a method described previously (Evans et al, 2015). For the bulk sort, 2 × 10 7 cells were Based on high and low AF488-fluorescence intensity gated fractions, 2.5 × 10 5 cells were deposited into 5-ml collection tubes containing the culture medium.…”

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

“…Fluorescence‐activated cell sorting (FACS) is commonly used for cell cloning in cell line development, and has the advantage of being a high‐throughput method that can deposit single cells into wells of multiwell plates, meeting the regulatory demands for clonality and assess cellular characteristics at the single‐cell level. Combining FACS with postsorting visualization by fluorescent imaging showed that >99.5% of cells were clonal . Various flow cytometry‐based screening methods have been developed to enable selective isolation of high‐producing clones.…”