Controlling cell-based bioassay performance through controlled preparation of bioassayready
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1 Controlling cell-based bioassay performance through controlled preparation of bioassayready cells Teresa Surowy September 2013 Promega Corporation
2 Introduction The importance of cell-based bioassays and cells in bioassays Measuring biological activity of a biologic is the only means of quantifying activity of the tertiary or quaternary structure and is critical for biologic lot release Cell-based bioassays measure this biological activity in a mechanism of action assay Biological relevance In vivo animal model Cell-based bioassay Binding or biochemical assay Variability Cells are often the most variable component of the cell-based bioassay Promega Corporation 2
3 Knowing and controlling your cells 1) propagation Thorough characterization and consistency for bioassay precision and accuracy A. What do the cells look like at various stages of growth? Photograph. Use for cross training. Establish optimized disruption procedures for accurate counting and passage. o Do suspension cells clump or stick to TC surfaces? o Select disruption procedures for adherent cells that work best for cell counting and for bioassay. Promega Corporation 3
4 Knowing and controlling your cells 1) propagation Thorough characterization and consistency for bioassay precision and accuracy B. Establish counting protocols and programs (if automated) that accurately and reproducibly determine cell density and viability o For cell production and o For cells that are harvested for use in bioassay. o Be cell specific. Establish growth curve and doubling time. Establish and adhere to limits on cell volume / culture vessel surface area ratio. Promega Corporation 4
5 Knowing and controlling your cells 1) propagation Thorough characterization and consistency for bioassay precision and accuracy C. Establish seeding density and maximum density for propagation. Stay within log phase. Set a rigorous passaging schedule. Optimize centrifugation. o Set viability limits and limits for performance in bioassay. Establish stability of response to treatment (for assay response) over passage and/or stability of bioassay over passage of engineered cells. o Set your limits based on bioassay performance. On scale-up, ensure you characterize and adapt processes to generate equivalent cells. Promega Corporation 5
6 Knowing and controlling your cells 2) harvest, cryopreservation, vial filling and use in assay Consistency and thorough characterization for bioassay precision and accuracy A. Optimize centrifuge speeds at harvest for high viability and acceptable recovery. Optimize mixing speeds at dispense to avoid shear. Establish how long your cells can be in freezing medium before cryopreservation and still perform consistently in bioassay. Set the time limit for vial filling and handling. Use a controlled rate freezer and optimize the program for each cell type. Promega Corporation 6
7 Knowing and controlling your cells 2) harvest, cryopreservation, vial filling and use in assay Consistency and thorough characterization for bioassay precision and accuracy B. Establish counting protocols and programs (if automated) that accurately and reproducibly determine cell density and viability for cells used in bioassay. o Make them cell specific. Optimize thawing procedures for performance in bioassay. Do the cells settle relatively quickly? Consider trituration during assay pipetting and practice for consistency. Cross train on everything including in different labs where the assay will be run. Promega Corporation 7
8 Thaw-and-use bioassay-ready cells Promega Corporation 8
9 Cells as Reagents Thaw-and-use.faster & better Traditional Method: Assays using fresh cells from culture (~1-2 weeks) Thaw Cell culture Cell count, harvest, resuspend in assay buffer, plate for assay, assay Read plates Cells as reagents : Thaw-and-use (3-24hr) Faster Less variable More convenient Thaw-and-Use Resuspend in assay buffer, plate for assay, assay Read plates Ample cell banks Promega Corporation 9
10 Process control needs to be rigorous Levels of control: Processes, characterizations and specifications 1. Determine and optimize cell production and QC processes 2. Choose characterizations that appropriately measures process control for bioassay performance: Consistency, uniformity, ability to meet critical cell requirements 3. Choose optimized processes and repeat to set cell reagent specifications Promega Corporation 10
11 Critical process parameters for thaw-and-use cells Critical process parameters: Cell propagation and harvest must be characterized and wellcontrolled Vial filling must be optimized and consistent Vial freezing must be optimized and consistent Viability and cell density measurements must be optimized and cellspecific Cell performance upon thaw must be characterized and consistent Other specifications for cells should be established after characterization and optimization of QC processes Seed stock vial Propagation Cell harvest Promega Corporation 11
12 Fold of Induction Critical thaw-and-use cell reagent requirements Critical cell reagent requirements: Cells must be of high viability, retain that after thaw, and an acceptable viability range must be selected for use in bioassay Cells must be of consistent cell density with a specified range Cells should respond to assay stimuli consistently Cells must meet sterility, mycoplasma-free, identity, species homogeneity requirements Bioassay performance using the cells must be consistent batch to batch Cells should meet requirements for accurate and precise biologic potency determination in the appropriate range in the bioassay Relative potency 50% 100% 150% Log Log[control 10 [B1 antibody], antibody], g/ml g/ml Promega Corporation 12
13 Evaluated passage stability for impact on bioassay responses Passage stability of thaw-and-use cells were evaluated in the reporter bioassay for consistency in response Passage Fold induction EC 50 (g/ml) e e e e e e-009 Promega Corporation 13
14 Evaluated impact of cell harvest density on bioassay parameters Range for FI consistency Range selected Range for EC 50 consistency Harvest density EC 50 (g/ml) FI 1 1.0e e e e Consider all relevant parameters for bioassay performance Promega Corporation 14
15 Evaluated the impact of cell culture changes on bioassay responses to cell stimulation Overgrown Cells Show Altered Response to TNFα cell viability is very similar but bioassay response is different! Apoptosis (caspase 3) readout Cell Consistency is Key to Bioassay Consistency : Promega Corporation 15
16 Evaluated cell production process scale-up for impact on bioassay T-flask to roller bottle, maintenance of surface area to culture volume ratio Go ahead for scale up Culture vessel EC 50 (ng/ml) Fold induction T-flask RB trial RB trial Promega Corporation 16
17 Evaluated impact of time in freezing medium before cryopreservation Thaw-and-use cells for bioassay.... Promega Corporation 17
18 Evaluated time limits for consistency of cell response to ligand treatment in assay after thaw-and-use Cells in thaw-and-use format were thawed for assay and evaluated for consistency in assay performance with time in 96-well plate before cell stimulation treatment for bioassay Time after thaw 30 minutes 45 minutes 60 minutes EC50 (ng/ml) Hill slope Promega Corporation 18
19 Fold induction EC50 (ng/ml) Evaluated assay performance stability to vapor phase liquid N 2 storage of thaw-and-use cells 25 Four different lots of thaw-and-use cells for bioassay were evaluated in the bioassay for stability of the bioassay performance across several months. Both EC50 and fold induction were evaluated in the reporter assay Month X Month Y Month Z Promega Corporation 19
20 Characterization of impact of cell viability on bioassay performance of cells in thaw-and-use format Cells frozen in freezing medium were mixed with dead cells prepared by freezing in propagation medium to create a series representing % viabilities, and used in the bioassay. % viability EC50 (ng/ml) These results were used to set limits on viability of cells upon thaw for bioassay Promega Corporation 20
21 RLU Demonstration of cells in thaw-and-use format performing equivalently to those grown in continuous culture WIL2-S cells at 12.5 K per well in ADCC Reporter Bioassay 150, , ,000 75,000 50,000 25,000 Frozen cells Continuosly cultured cells Final Rituximab (ng/ml) Effector cells: FcgRIIIa/NFAT-luc2 engineered Jurkat effector cells (thaw-and use); Ab: Rituximab dilution series; Induction time: 6 hours Promega Corporation 21
22 Cells as Reagents (Thaw-and-Use): Complete QC tests with specifications Cell doubling time under propagation conditions Cell density Cell viability after thaw Fill volume Mycoplasma (Hoechst) Mycoplasma (Direct culture) Sterility USP <71> STR analysis cell ID profile (human) CO1 analysis (cytochrome oxidase) test for presence of species (human and other potential contaminants) Bioassay performance (EC 50 and fold induction or response) 22 Promega Corporation 22
23 THANK YOU! Promega Corporation 23
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