1. Optimization of high-cell density cultivation to produce monoclonal antibody in glycoengineered Pichia pastoris by real-time monitoring of glycerol and methanol Sehoon Kim Merck & Co., Inc 3rd International Conference on Bioprocess and Biosystems Engineering September 14, 2015

2. Slide 2 Contents  Introduction: Glycoengineered Pichia pastoris and its bioprocess  Off-line measurement for substrates  Real-time measurement  Antibody production in platform bioprocesses  Process analyses in real time  Optimization of methanol dose  Conclusions  Acknowledgement

3. Slide 3 Glycoengineered Pichia pastoris Late Golgi Hamilton et al., Curr Op Biotechnol, 2007

4. Slide 4  Strategies to maximize the productivity Increasing specific productivity (q p (t)) Increasing biomass (XV(t)) Increasing induction time (t f )  Constraints Mass transfer rate (e.g., OUR) Cell fitness (minimize X d ) and media Protein quality (e.g., protein intactness, glycosylation) High-cell density fed-batch culture of P. pastoris with pAOX1

5. Slide 5 I II III Batch Fed-Batch Induction 1 o carbon Inducer Glycerol - Glycerol - Methanol High-cell density fed-batch culture of P. pastoris with pAOX1

6. Slide 6 Conventional method: Off-line data for glycerol and methanol  Off-line measurement at random time-points When to be zero? Discrete measurement only No clues of methanol profiles Sample numbers affect culture volume

7. Slide 7 On-line method: Real-time analysis with Bioprocess monitor Near Infra-red (NIR): RTBio-1000 model (ASL Analyitcal) Kim et al., Biochem Eng J, 2015

8. Slide 8 High-cell density fed-batch culture of P. pastoris  Methanol-limited method -Exponential feed of methanol -[MeOH] ~ 0  Oxygen-limited method -MeOH dose @ DO-spike -OUR (mmol/L/h) control Kim et al., Biochem Eng J, 2015

9. μ max > 0.1 /h μ Gly = 0.08 /hμ MeOH = 0.0063 /h BatchFed-Batch (Gly) Glycerol Glycerol/MeOH pathways in P. pastoris – Carbon flux changes before and after induction DAS1 FLD1 FDH1 Peroxisome Fed-Batch (MeOH) MeOH Slide 9

10. MeOH FAD FADH 2 O2O2 H2O2H2O2 ½ O 2 + H 2 O AOX CAT Form CH 3 OH H 2 CO Peroxisome Dissimilation Assimilation MeOH-limited Normoxic (20% DO) MeOH FAD FADH 2 O2O2 H2O2H2O2 ½ O 2 + H 2 O AOX CAT Form CH 3 OH H 2 CO Peroxisome Dissimilation Assimilation Oxygen-limited Anaerobic – Hypoxic (< 6% DO) High energy demand Gluconeogenesis activation Fatty acid accumulation Nitrogen depletion OUR optimization is required Longer induction is achievable Biomass Titer Biomass Titer NADH Metabolomic fluxes of methanol utilization pathway in two different fed-batch processes Kim et al., J Biotechnol, 2013 10 Slide 10

11. Slide 11 On-line method: Real-time analysis with Bioprocess monitor Powerful and accurate analyses of glycerol and methanol in Pichia pastoris process Exact measurement of growth phase transitions: Batch  Fed-batch  Induction Methanol profile: Peak-to-peak interval, Max and Min, Slopes Kim et al., Biochem Eng J, 2015

12. Slide 12 Feed weight DO Fo1 (Glycerol pump) Glycerol Batch phase On-line parameters

13. Slide 13 Glycerol fed-batch phase Fo2 (Methanol pump) Methanol

14. Slide 14 Induction phase Fo2 (Methanol pump) Methanol DO

15. Slide 15 Fo2 (Methanol pump) Methanol DO Induction phase

16. Slide 16 Feed weight DO Fo1 (Glycerol pump) Glycerol Fo2 (Methanol pump) Methanol Induction phase Kim et al., Biochem Eng J, 2015

17. Slide 17 2.5g/LMeOH dose 5g/LMeOH dose 10g/LMeOH dose 20g/LMeOH dose30g/LMeOH dose Analysis of methanol consumption rates Kim et al., Biochem Eng J, 2015

18. Slide 18 Optimization of methanol dose  10g/L methanol dose resulted in high titer of mAb for 125h of induction Kim et al., Biochem Eng J, 2015

19. Slide 19 Conclusions  High cell density culture of Pichia pastoris was achieved by fed-batch methods, methanol-limited and oxygen-limited methods  Two substrates, glycerol and methanol were accurately monitored in real-time using on-line bioprocess monitor  The monitor tracked critical substrate conditions such as peak concentration, depletion, and consumption rate  The antibody titer in glycoengineered P. pastoris was highest at 10 g/L methanol dosages in oxygen-limited fermentation  Bioprocess monitor was robust enough to continuously operate over 365 days with no recalibration

20. Slide 20 Acknowledgement Adam Nylen Ishaan Shandil Muralidhar Mallem Erik Hoyt Jiang Bo Marc d’Anjou Natarajan Sethuraman Kaylee Lanz Christine Evans Elizabeth Gibson Jonathon Koerperick Daniel Cooley Gregory Brower Gary Small Mark Arnold ASL Analytical Inc Merck & Co., Inc

21. Slide 21 Thank you! Questions? sehoon_kim@merck.com