Advanced® 20G
8Pages

Evaluation of The 20G — A 96-Well Format High Throughput Osmometer Louis Cheung2, Roy Damiani1, Mark Kacprowicz1, Martin Gawlitzek 2, and Shun Luo2 Advanced Instruments, Inc., Norwood, MA 02062 Late Stage Cell Culture Process Development, Genentech Inc., South San Francisco, CA 94080 1 Acknowledgements Advanced Instruments: John Coughlin, Peter Costas, Pete Emond Genentech: Bob Kiss, Christina Bevilacqua, Robb Shawley, Sonali Nigam AbstracT An automated high throughput osmometer was developed in collaboration between Advanced Instruments and Genentech. The automated osmometer, 20G, combines state-of-the-art osmometry and robotics technology. The 20G can process samples in 96-well format in 35 minutes with the same accuracy as current standalone osmometers (i.e., under 1% CV for samples between 50 and 900 mOsm/kg H 2O). To correct for the evaporation that arises from open lab humidity when measuring 96 samples, a correction constant was established. Corrected osmolality was found to be consistent when using osmolality standards. The 20G performed within specification when used to measure cell culture media with and without sample preparation. The automated osmometer is designed to support a scaledown high throughput cell culture process development platform developed at Genentech. Introduction Most current osmometers use freezing-point depression technology to measure osmolality, which represents the total solute concentration of an aqueous solution. Osmolality information is very important to the biotechnology research and manufacturing industry due to its relationship between cells and media. The automated 20G Osmometer combines state-of-the-art osmometry and robotics technology to process samples in 96-well format with the same accuracy as current standalone osmometers. The automated osmometer is designed to support a scale-down high throughput cell culture process development platform developed at Genentech.

Functional Specifications • Sample Volume: 20 μL • 96-well Format: 12 x 8 layout (compatible with SBS guidelines) • Throughput: 96 samples in 35 minutes • Calibrated Range: 50 to 850 mOsm/kg H 2O with ±1% accuracy • User Interface: Software friendly and comparable to other state-of-the-art laboratory automation software • Data Output: Compatible with Biomek® FX software • Regulatory Approvals: Certified to applicable safety and EMC standards System Status Light Operator Access Portal Safety Interlocks Osmometer Modules Keyboard & Mouse Rack Positioning Fixture Computer

Analysis of Osmolality in 96-well Format • 290 mOsm/kg H 2O Reference Solution − Manufacturing Spec: 290 ± 2 mOsm/kg H 2O − Total Run Time: 31 minutes Manufacturing Specification: 290 ± 2 mOsm/kg • Additional measurements within osmometer enclosure: − Temperature: 28.1°C − Relative Humidity: 33% Potential Problem and Resolutions • Osmolality indicates a gradual upward trend during the 96-well format analysis due to sample evaporation • Averaged osmolality of the last eight samples (Sample #89 to #96): ~ 298 mOsm/kg H 2O Solutions • Approach 1: Control temperature and humidity within the...

Mathematical Models % Increase in Osmolality Relative to the Initial Measured Reading Adjusted Osmolality (mOsm/kg H2O) Determination of Correction Constant Plot: % increase in osmolality (relative to the initial measured reading) versus time, where % increase in Osmolality = [(Experimental Value – Initial Measured Value)/Initial Measured Value] x 100% Time (mins) Use of Correction Constant Adjust the experimental values using the correction constant, where Correction Constant = Slope of the Plot (% increase in osmolality versus time) = 0.0885% increase in osmolality per minute Adjusted...

Results indicated that Correction Constant: • Varies from 0.077 to 0.122 % increase in osmolality per minute • Seems to be dependent on humidity - Correction constant decreases as humidity increases • Seems to be independent of concentration of standards - At 32% humidity, Pair Student's t-test shows that the 100 and 500 mOsm/kg H20 standards possess a comparable correction constant (p > 0.8293) • More experiments are required over a broader humidity range to confirm findings and to understand the change of correction constant with respect to humidity. ASSAY SPECIFICATIONS Measurements of...

Additional Experiments 12 Osmolality — With Cells Osmolality — Without Cells Viable Cell Density (x 106 cells/mL) Osmolality of cell culture supernatant (with cells vs. without cells) Osmolalities of cell culture supernatants in the presence (N=3) and absence (N=3) of cells were measured. Results indicated that cell mass in cell culture supernatant sample does not interfere with osmolality measurement. Averaged Osmolality (290 mOsm Ref) % CV Averaged Osmolality of 290 mOsm Reference (mOsm/kg H2O) Effects of sample volume Manufacturer’s suggested sample volume: 20 μL Results indicated that...