Sehoon Kim Merck & Co., Inc 3rd International Conference on Bioprocess and Biosystems Engineering

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

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

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

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

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. 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. FDH1 DAS1 FLD1 Glycerol/MeOH pathways in P. pastoris – Carbon flux changes before and after induction Batch Fed-Batch (Gly) Fed-Batch (MeOH) μGly = 0.08 /h μMeOH = 0.0063 /h μmax > 0.1 /h MeOH Peroxisome Peroxisome Glycerol

10. Metabolomic fluxes of methanol utilization pathway in two different fed-batch processes Oxygen-limited Anaerobic – Hypoxic (< 6% DO) MeOH-limited Normoxic (20% DO) MeOH MeOH Peroxisome Peroxisome NADH NADH FAD FADH2 FAD FADH2 Dissimilation Dissimilation Form MeOH Form MeOH CH3OH H2CO CH3OH H2CO AOX Assimilation AOX Assimilation O2 H2O2 O2 H2O2 Biomass Biomass CAT CAT Titer Titer ½ O2 + H2O ½ O2 + H2O • OUR optimization is required • Longer induction is achievable • High energy demand • Gluconeogenesis activation • Fatty acid accumulation • Nitrogen depletion Kim et al., J Biotechnol, 2013 Slide 10

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

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

14. DO Fo2 (Methanol pump) Methanol Induction phase

15. DO Fo2 (Methanol pump) Methanol Induction phase

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

17. Analysis of methanol consumption rates 2.5g/LMeOH dose 10g/LMeOH dose 5g/LMeOH dose 20g/LMeOH dose 30g/LMeOH dose Kim et al., Biochem Eng J, 2015

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

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