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

Abstract

Generating a highly productive cell line is resource intensive and typically involves long timelines because of the need to screen large numbers of candidates in protein production studies. This has led to miniaturization and automation strategies to allow for reductions in resources and higher throughput. Current approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. New nanofludic technologies offer novel solutions to surpass these limits, further miniaturizing cell culture volumes (10⁵ times smaller) by growing cells on custom nanofluidic chips. Berkeley Lights' OptoElectro Positioning technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Using a fully integrated technology platform (Beacon), common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip. Here, we describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that commercial production Chinese hamster ovary cell lines can be isolated, cultured, screened, and exported at high efficiency. We compare this process head to head with a FACS-enabled microtiter plate-based workflow and demonstrate generation of comparable clonal cell lines with reduced resources. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1438-1446, 2018.

Keywords: Cell line development; automation; nanofluidics.

Figure 1

Assessment of workflow efficiency of the Beacon platform. (A) Representation of workflow with images of cells and colonies. (B) Number of pens that contain empty, single, or multiple cells per chip after loading operation. Percentage denotes highest and lowest percent singles loaded. (C) Cell count of pens after 3 days from single cell loads. Percentage denotes number of single clones with positive cell growth. (D) Counts of unpenned cells after export workflow. Percentage denotes numbers of pens with exports greater than three cells. (E) Count of wells with growth after export.

Figure 2

Comparison of a microtiter plate‐based cloning workflow vs. a nanofluidic chip subcloning workflow. A depiction of the steps involved in performing a clonal isolation and expansion workflow using two approaches. Differences are highlighted in boxes for FACS‐based workflow(solid) and Beacon workflow (dotted).

Figure 3

Correlation of Spotlight Titer analysis of early clones with fed‐batch specific productivity of mature clones. (A) Secretion and growth measurements measured on OptoSelect™ chips. The Spotlight secretion assay generates a ranked assay score. (B) Growth assessment on chip is performed through performing cell counts and calculation of doubling time. Selected clones with a wide range of growth and secretion highlighted in white. (C) Scatter plot of individual clones analyzed early on the Beacon using Spotlight Assay (X‐axis‐Titer score) and analyzed 50 days later in a suspension fed‐batch production experiment (Y‐axis‐Relative specific productivity).

Figure 4

Comparison of clonally derived cell lines from FACS‐based and Beacon Workflows. (A) Fed‐batch screening of mature clones. Clones from both methods screened using a fed‐batch production screen and accumulated titer measured (Normalized grams/liter). Top clones from each method highlighted with open circles. (B) Bioreactor screening of top clones. Top 3 Beacon clones (Black) screened with top 3 FACS‐based clones (white). Plot of VCD (Top Left), viability (Top Right), accumulated titer (Bottom Left), and specific productivity (Bottom Right) over process duration.

Figure 5

Assessment of stable cell lines on the Beacon platform. (A) Interrogating cell growth from single cells (blue) vs. shaken suspension pools (orange). (B) Single clone analysis of stable populations for growth and secretion. Plot of cell numbers after 3‐day growth vs. spotlight secretion assay score. (C) Specific productivity profiles of five stable pools after culturing.