Microfluidic chip‐based single‐cell cloning to accelerate biologic production timelines

Abstract

Cell line development (CLD) represents a critical, yet time-consuming, step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence-activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared with standard process-derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability.

Sorting single T cells based on secreted cytokines and surface markers using hydrogel nanovials

“…Here the bottleneck for throughput becomes the flow cytometer event rate 22 . Other microfluidic platforms have been limited to a few thousand cells 23 or a maximum of 50,000 cells per run 15,20 . The current throughput can be further increased by reducing the size of the nanovials and decreasing the nozzle size of the FACS machine to process nanovials at a rate that could approach 10,000 events per second.…”

“…All other secretion assay steps can be completed with general lab supplies such as pipettes, tubes, and centrifuges. Other microfluidic approaches to analyze secretions, such as the Berkeley Lights Beacon system 15,20 or Isoplexis Isolight system 8,21 require a specialized instrument, which are not as widely available as FACS. We also demonstrate the ability to perform multiplexed surface and secretion-based sorting with <1% crosstalk, leveraging the localized cytokine capture sites on the nanovials.…”

Challenges and Emerging Technologies in Biomanufacturing of Monoclonal Antibodies (mAbs)

“…In terms of bioprocessing, CHO cells are robust and are easily adapted to grow in suspension in chemically defined and serum-free media making them readily amenable to process scale-up. CHO cell line development has generated cells with specific productivities (Qp) in the range of 5-20 pg/cell/day [22] and advances in single cell sequencing could increase this further [23]. Other mammalian cell lines like baby hamster kidney (BHK) cells and murine lymphoma cells lines such as NS0 and Sp2/0 also provide human-like glycosylation patterns but productivity levels can be significantly lower [24].…”

Application of microfluidic technology in antibody screening

“…Some commercially available platforms, such as Beacon platform from Berkeley Lights, [ 103 ] CellCellector platform from XenoMouse, [ 104 ] and Cyto–Mine. [ 105 ] can also offer single‐cell screening instruments for antibody discovery.…”