these difficulties were overcome by expanding cells to a passage level that maximized yield while retaining endothelial characteristics, and generating a large bank of ready-to-use cryopreserved cells for an assay to measure the potency of a therapeutic anti-VEGF monoclonal antibody by AlamarBlue reduction. For each assay, a vial of cryopreserved cells is thawed and resuspended in assay medium, and then directly dispensed in the assay microplate. Te authors note that it is critical to identify optimal growth and freezing media, cell passage number, freezing and thawing conditions, centrifugation speeds and cell density in vials, and that some cell types are particularly sensitive to thawing conditions.
Working cell banks of about 800 vials were created, and reproducibility of bank preparation by different facilities was demonstrated. Commercial vendors were then identified to generate large banks, with qualification performed by the originating laboratory. It was found that the use of ready-to-use cells significantly improved method transfer, avoiding the variability due to cell culturing which often proves a major challenge in method transfer between facilities.
Commercially available ready-to-use cells and assay kits
While some biopharmaceutical companies choose to develop banks of their own proprietary ready-to-use cells, there is also the possibility of purchasing commercially available ready-to-use cells in batch sizes and with supporting methodology, technology and certification as appropriate to the intended use. Commercially available ready-to-use kits for specific therapeutic targets are becoming increasingly prevalent and provide a solution to jump start method development for potency and characterization assays. Tese ready-to-use kits can provide all required reagents (ready-to use cells, plating reagents, control ligands) and detailed standard operating procedures to allow easy implementation of the assay in a QC lab.
An example of such a system is the PathHunter® Bevacizumab assay (DiscoverX) which supports development of biosimilars to anti- VEGF therapeutics (e.g., Bevacizumab). Te mechanism of action of these agents is to inhibit the growth of blood vessels in tumors by binding to specific VEGF isoforms, preventing activation of the primary VEGF receptor found on endothelial cells, VEGFR2. Te PathHunter® Bevacizumab assay quantifies VEGF-A-induced homodimerization of the VEGFR2 receptor, which is the first step in the activation cascade of the receptor. Tis activation or dimerization event is blocked by the presence of anti-VEGF-A antibodies like Bevacizumab. Importantly, this highly robust method of testing Bevacizumab potency produces equivalent IC-50 values to the long- standing HUVEC proliferation assay with better precision and accuracy, and in a fourth of the time required to run the legacy assay [4].
Te ready-to-use cells are manufactured to defined specifications for low intra-lot and inter-lot variation, and are shipped with a Certificate of Analysis. All reagents in each kit have been optimized to work together to achieve optimal performance in the assay, and the optimized protocols have been qualified to demonstrate repeatability, precision, accuracy and linearity utilizing a relevant therapeutic agent for the drug target.
As therapeutics for ever more difficult drug targets move towards the clinic, the potency assays to support them are also becoming increasingly more complex. For certain immuno-oncology targets, such as the immune checkpoint inhibitor PD-1, the use of co-culture assays to reflect the complex physiology of the target are required. For such targets, the use of frozen ready-to-use cells are even more important to minimize the variation that would be inherent in utilizing two different cell types in one assay. Examples of such assays are the PD-1 bioassays produced by Promega and DiscoverX, that use one cell type to express the PD-1 receptor (e.g., Jurkat cells) and quantify receptor engagement (or blocking with a therapeutic agent), and a second cell type to express ligand and/or stimulate activation of the assay. Both bioassays are robust, precise and accurate, suitable for use in a QC environment.
Ready-to-use cells represent one of the major recent advances in bioassays for potency and characterization of biopharmaceuticals. Te bioassay has traditionally been viewed as one of the most problematic of the analytical techniques required for QC and batch release. Frozen ready-to-use cells offer improvements in use of laboratory resources, assay scheduling, robustness and assay variability. Moreover, the increasing availability and variety of ready-to-use kits will allow more extensive investigative studies of biopharmaceuticals by avoiding the lengthy and costly development required for establishing a cell-based assay in a laboratory.
References
1. Menendez AT, Ritter NM, Goyal J, Jani D, Zmuda J. Recommendations for cell banks used in GXP assays: preparation, characterization, and storage. Bioprocess Int. 10(1), 26–40 (2012).
2. Burns CJ, Silva MM, Gray E, Robinson CJ. Quantitative RT-PCR as an alternative to late-stage bioassays for vascular endothelial growth factor. J. Pharm. Biomed. Anal. 47(3), 460–468 (2008).
3. Gazzano-Santoro H, Chan LG, Ballard MS, Young JC. Ready-to-use cryopreserved primary cells a novel solution for QC lot release potency assays. Bioprocess Int. 2(2), 28–39 (2014).
4. Lamerdin J, Daino-Laizure H, Saharia A, Charter NW. Accelerating biologic and biosimilar drug development: ready-to-use, cell-based assays for potency and lot release testing. Bioprocess Int. 14(1), 36–44 (2016).
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