1. Dedicated Analytical Solutions Near-Infrared Spectroscopy as a Process Analytical Technology in the Biotech Industry Presented to the Advisory Committee for Pharmaceutical Science Food and Drug Administration April 13, 2004 By Robert A. Mattes Foss

2. Dedicated Analytical Solutions Near-infrared Spectroscopy Spectral Region UV Visible Near-Infrared Mid-Infrared 2500 4,000 Functional Group Analysis Constituent Assay Appearance Elemental 700 14,285 400 25,000 200 50,000 25,000 nm 400cm-1

3. Dedicated Analytical Solutions Pharmaceutical Manufacturing NIR Applications Incoming Raw Materials Material Dispensary In-Process: Fermentation and Cell Culture Monitoring Warehouse Shipping Lyophilization

4. Dedicated Analytical Solutions BioPharm Process Reactor Control feedback On-line monitoring of critical process parameters Process control Raw Materials Process output Closed loop control (Real-Time) Temperature Sparging NIR monitoring Nutrient levels Control Capabilities with NIR/PAT Moisture level Melt detection In Lyo Process Improvement

5. Dedicated Analytical Solutions OrganismSupplierProductFluid BehaviorAnalyte (s) Escherichia coli (unicellular) Eli LillyBST (bovine somatotropin) Non-viscous Newtonian biomass, substrates, acetate (by-product), product Streptomyces fradiae (filamentous) Eli LillyTylosinViscous non- Newtonian biomass, substrates, product Pichia pastoris (unicellular) British Biotechnology, PLC Human ProteinViscous Newtonian biomass, substrates, product CHO-K1 (animal cell-line) British Biotechnology, PLC. Human ProteinNon-viscous Newtonian Biomass, substrates, product Industrial Organisms Studied NIR Bioprocess Method Development

6. Dedicated Analytical Solutions NIR Spectral Acquisition and Analysis for Bioprocesses

7. Dedicated Analytical Solutions NIR Spectral Analysis Analytical Region for Key analytes Second-derivative of Log(1/T) Glycerol, acetate, biomass t=0, t=27 hr

8. Dedicated Analytical Solutions NIR Bioprocess Trends Chart

9. Dedicated Analytical Solutions NIR Bioprocess Analytical Results

10. Dedicated Analytical Solutions Near IR applied to Mammalian Cell Cultivation  Industrial CHO-K1 cell line  In-situ NIR using transflectance measurements (1.2 mm pathlength)  2 Liter bioreactor run for 168 –240 hours, including a temperature change  Developed NIR calibrations for:  Glucose  Glutamine  Lactate  Ammonia Ref: SA Arnold, J Crowley, N Woods, LM Harvey, and B McNeil, Biotech and Bioeng 84(1), 13-19 (2003).

11. Dedicated Analytical Solutions Direct In-Line Analysis in Mammalian Cell Cultivation  Direct fermenter interface  Standard Ingold ports  Optimized for specific analytes/matrices  Dedicated analysis on defined media  Simultaneous results <1 minute  Monitoring and closed-loop control  Pilot plant/scale-up operations and manufacturing Direct Sample Interface

12. Dedicated Analytical Solutions Summary of NIR Results for CHO cell work AnalyteRangeSECR2R2 SEP Ammonia0.5- 2.0mM 0.0320.990.036 Glucose0.7-3.5g/L0.070.990.07 Lactate0.3-1.2g/L0.0080.990.014 Glutamine2-6mM0.120.990.31 Multifactor PLS calibrations developed on second derivative spectra with 217 samples

13. Dedicated Analytical Solutions Enlarged View of Second Derivative Spectra Used for Analysis of Lyophilized Product

14. Dedicated Analytical Solutions  NIR offers the advantage of real-time analysis in biotech sterile environments and the possibility of closed-loop feed back process control  Benefits in process …  Non-invasive analysis  Process optimization  Waste reduction  Better understanding of processes and  Safer, improved, more consistent product NIR Benefits for the PAT Biotech Initiative