Genetic Engineering & Biotechnology News

DEC 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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18 | DECEMBER 2018 | Genetic Engineering & Biotechnology News | GENengnews.com Frank F. Craig, Ph.D. Antibody-derived biologics have become a major class of modern medicine, particularly in the fight against cancer and autoimmune diseases. Crucial to the successful translation of antibodies into therapies are highly efficient antibody discovery and cell line development pipelines. The challenge for biopharma is to screen large cell populations for productivity, antigen specificity, or other parameters, and then isolate rare cells with assured clonality. Traditionally, this was achieved by the re- source-intensive method of limiting dilution, and more recently aided by semiautomated technologies such as fluorescence-activated cell sorting (FACS), colony picking, and cell- in-well imagers, but these offer only partial solutions for automated workflows. High- throughput cell-sorting methods cannot readily measure secreted protein, and current secreted protein screening methods are lim- ited in their ability to handle large numbers of cells. As a result, the screening, cloning, and verification steps for antibody discov- ery and cell line development still require manual intervention at each stage, creating substantial bottlenecks. Next-Generation Single-Cell Analysis Picodroplet cell encapsulation technology is a unique approach to single-cell analysis that can deliver higher throughputs, rapid yet gentle cell processing, single-cell dispensing to microplates, and monoclonality verification by imaging, in a fully automated platform. Sphere Fluidics' Cyto-Mine ® Single Cell Analysis and Monoclonality Assurance System is a next-generation single-cell analysis plat- form that integrates antibody discovery pro- cesses using patented picodroplet technology to encapsulate single cells, which can then be assayed and sorted automatically (Figure 1). 1) Cells encapsulated into picodroplets: The target cell population is prepared in preferred culture medium supplemented with an ap- propriate animal-origin-free (AOF) antibody- based detection reagent for the selected secre- tion assay. The cell suspension is then gently pumped through microfluidic channels and mixed with a biocompatible surfactant, which encapsulates a single cell (or pools of cells) in each 300-pL droplet of culture medium. 2) Incubation and secreted protein assay: Approximately two million generated pico- droplets are collected together into a chamber and incubated in situ at 37°C to activate the required assay. The assay could be used to detect secreted proteins, biomarkers, intracel- lular fluorescence (such as a viability stain), or an intrinsic functional assay marker. 3) Sorting positive cells: The picodroplets are sorted by fluorescence detection and gat- ing, similar to typical flow cytometry, with positives being actively channeled for col- lection. The population of cells selected for collection can be defined and adjusted ac- cording to each specific experiment. Selected positive picodroplets are stored in a chilled microchamber prior to dispensing, and nega- tive picodroplets that contain low-fluoresc- ing cells, or are empty, are diverted to waste. 4) Visual verification and dispensing: After completion of the sorting phase, positive pico- droplets are selected, imaged, and dispensed to individual wells of a 96- or 384-well micro- plate prefilled with preferred culture medium. The imaging process uses an ultra-high-speed brightfield camera to acquire multiple frames of each picodroplet, providing unambiguous visual evidence of a single cell progenitor. FRET-Based Protein Secretion Assays Förster resonance energy transfer (FRET)- based assays are used in the Cyto-Mine platform to detect protein(s) secreted by en- capsulated cells. The assay can be custom- ized by designing AOF fluorescent detection probes specific for the protein of interest, such as IgG (e.g., for a productivity screen) or antigen-specific IgG (e.g., for a hybrid- oma fusion screen or B-cell mining). When the secreted protein is recognized by the de- tection probes, a three-body FRET complex is formed, which induces a FRET-mediated shift in fluorescence. This signal is used to determine the quantity of secreted protein and select picodroplets for collection. IgG Secretion Assay for Cell Line Development Sphere Fluidics developed an assay to with complex automated valve sequences. Although the potential for bag leaks is a dis- advantage, this exigency can be dealt with through adoption of a pre-use pressure test after bag installation. She adds that SUMs have an advantage over SUBs in that generally bag leaks from a SUM have less potential impact to product quality and batch success than leaks from SUBs. Leitch also mentions bioburden control and the risk of microbial contamination in single-use technology. The economic impact of batch failure due to a bioburden incident is enormous, and could cost a firm billions of dollars in lost revenues and correction of the root causes. A significant advantage of single-use bioreactors is that integrity is- sues of the sterile boundary are highly vis- ible. From a bioburden control perspective, since there are no reusable product contact surfaces in single-use systems, there are no risks of biofilm development which reduces potential multi-lot product quality risks. Balancing Options In choosing between single-use and multi- use solutions, there are a number of factors that need to be reckoned with, as companies seek to develop a set of general principles to govern the selection process. Today, the over- riding consideration is the scale of the opera- tion, with decision makers favoring using sin- gle-use technology in batches under 1000 L and multi-use solutions for larger quantities. Other factors affecting the decision include the type of product (batch versus continu- ous), the phase (clinical or research) of the product, reductions in cleaning requirements, capital investment, turnaround time, and risk of product cross-contamination. The weight of these considerations is sure to change in the future, as the technology is changing rap- idly, which is sure to influence the decision. The Future of Antibody Discovery and Cell Line Development Single-Use Systems Continued from page 17 Bioprocessing Tutorial Sphere Fluidics Integrates Single-Cell Screening Processes to Identify Rare and High-Producer Clones Figure 1. Schematic of the automated Cyto-Mine® workow, which integrates cell screening, sorting, and monoclonality assurance by imaging into a single platform using picodroplet technology. Figure 2. Cyto-Mine IgG secretion assay. (A) IgG-specific probes are trapped within each picodroplet and bind to secreted IgG to form a three-body FRET complex. (B) Scatterplots of FRET signal for picodroplets containing the indicated concentrations of human IgG with IgG detection probes. (C) Standard titration curve derived from data in B. (D) Scatterplot generated from picodroplet-encapsulated CHO cells incubated with IgG-specific detection probes. B C D

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