Mixing, mass transfer and bioprocess scaling up and down in new generation single-use bioreactors. Nico Oosterhuis CELLution Biotech

Transcription

1 Mixing, mass transfer and bioprocess scaling up and down in new generation single-use bioreactors Nico Oosterhuis CELLution Biotech

2 Questions of today Mixing and mass transfer in single use bioreactor: can we design a multipurpose single-use bioreactor? Can it be scaleable?

3 Why Single use? STERILITY Reduced cross contamination Pre-sterilized (validated) Easy sterile connections COSTS No cleaning Simple validation Simple infrastructure Lower maintenance Flexibility turn-around

4 Reasons to apply single-use What is the pharma-industry telling us today? Source: Paul Cook, 2010

5 Many types of single use bioreactors (Eibl, 2008)

6 Applicability today s single-use bioreactors? Rocking systems Stirred systems Mass transfer, k l a max = hr -1 Restricted heat transfer Scaleability present (rocking) systems questionable Only applicable for mammalian cell culture

7 Single-use bioreactors applicability? REACTOR TYPE kla VOLUME TYPE OF BAG TYPE OF MIXING SUPPLIER (hr -1 ) WORKING Wave Bioreactor L pillow shape rocking GE Healthcare <30 Cultibag RM L pillow shape rocking Sartorius Stedim Biotech < 30 AppliFlex 1 25 L pillow shape rocking Applikon Biotechnology < 40 CultibagSTR L tankliner stirred Sartorius Stedim Biotech <40 Thermo-Fischer SUB L tankliner stirred (Hyclone) < 40 XDR L tankliner stirred XCellerex < 20 Nucleo L square 3D Paddle agitation ATMI / Pierre Guerin < 20 Shaking bioreactor < 200 L tankliner orbital shaker Kühner/ExcellGene < 40 CellMaker Regular 1 50 L bubble column rotating sparger Cellexus < 10 => All single-use systems limited applicable

8 Mammalian vs microbial in biopharma? MAb Enzymes Hormones/GF Toxin-conjugates Scaffolds Therapeutic proteins Insulins Protein vaccines PEG-Conjugates MAb Mimetics Phase III projects in 2009 (source: Boehringer 2011) Microbial Mammalian 44% 56%

9 Single use bioreactors today Rocking type: mainly applied on small scale (< 150Ltr) Stirred type: up to 2000 Ltr All: Mixing, mass- and heat transfer restricted Only applied for mammalian processes Need for high-performance and microbial processes

10 Regime analysis (Kossen & Oosterhuis, 1985) Regime analysis: comparison of typical time constants t 0,0001 0, (SEC) (MIN) (HR) MASS ACTION ENZYME INDUCTION SELECTION ALLOSTERIC INTERACTION ORGANISMS MIXING MASS TRANSFER REACTOR DYNAMICS REACTOR/ENVIRONMENT

11 Regime analysis microbial TRANSPORT STR STR Wave CT r CT r < 10L > 100L 10L 100L Oxygen transfer < 10 (s) < 15 (s) > 90 (s) (s) (s) Mixing < 5 (s) (s) > 30 (s) < 10 (s) (s) Heat transfer (s) (s) n.a (s)? CONVERSION O2 consumption 6 15 (s) Heat production (s) Substrate conv. Growth > 1000 (s) > 2000 (s)

12 Regime analysis cell-culture TRANSPORT STR STR Wave CT r CT r < 10L > 100L 10L 100L Oxygen transfer (s) > 200 (s) > 150 (s) (s) (s) CO2 transfer > 500 (s) > 500 (s) > 250 (s) (s) (s) Mixing (s) (s) > 60 (s) (s) (s) Heat transfer (s) (s) n.a (s)? CONVERSION O2 consumption > 200 (s) CO2 production Heat production < 200 (s) p > 800 (s) Substrate conv. Growth hours days

13 Required mass- and heat transfer Cell culture Microbial culture (E.coli) OUR: 0,2 2 mmol/10 6 c/hr OUR: 4 mmol O 2 /g.hr When OUR = OTR When OUR = OTR 20 x 10 6 c/ml => 4 mmol/l.hr 50 g/l => 200 mmol/l.hr 1 4 mmol / l.hr => k l a 20 hr -1 (air) 200 mmol/l.hr => k l a 1000 hr -1 (air) 200 mmol/l.hr => k l a 400 hr -1 (50% O 2 ) 1 Cooling: 469 KJ/mol O 2 consumed At 10L => 8 W cooling power needed MASS TRANSFER might be an ISSUE Cooling : 469 KJ/mol O 2 consumed At 10L => 260 W cooling gpower needed MASS and HEAT TRANSFER is an ISSUE

14 2-Dimensional movement in rocking motion Increase of mass transfer due to additional translational movement

15 Stirring versus 2-D shaking? The microenvironment of the cell determines its physiological behavior. Conditions have to be compared: local concentrations of O 2 and CO 2 nutrients energy dissipation scale (shear) time constants t gradients

16 Mixing and mass transfer Shape/geometry of the bioreactor or power / volume?

17 Stirring versus 2-D shaking GAS phase LIQUID phase Mass transfer O 2 CO 2 O 2 CO 2 GRADIENTS Nutrients mixing CELL ph Temperature SHEAR FORCES Membrane transport Side-products Product

18 Design using an engineering view MASS-transfer & mixing Real SURF motion (two dimensional i rocking) )improves mixing i and oxygen transfer Proper and accurate temperature control Heating by convection (incubator) Direct cooling (heat exchanger under the bag) Short mixing times => no gradients EASY handling & real single use Bags flexible in volume Robust one box equipment (stackable) In-line measurements non-invasive, fully disposable (ph, DO,), bottom mounted, others (glucose/lactate) Scaleable Same process performance at different scales

19 RESULTS Mass transfer E.coli Heterotrophic t algae Pre-culture Corynebacterium (lysine) Mammalian cells (PER.C6 - CHO) Scaleability

20 Mass-transfer CELL-tainer

21 Mass transfer results (dynamic, water, 20 o C) 800 At higher rocking rate: decrease of k l a due to inertia of the liquid kla (hr-1) 700 CT'r (10L) 600 CTr (15L) 500 Wave (20-10L) Range microbial (35 55 rpm; k l a >300 hr -1 ) rocking speed (rpm) / at maximum circle (angle) Range cell culture operation (15 25 rpm; k l a hr -1 )

22 Comparison to STR 0,25 0,2 (s-1) Kla 0,15 0,1 CELL-tainer STR 0, P/V (W/Ltr)

23 Mass transfer volume rocking speed Interdependence k l a volume rocking speed

24 Linear evolution k l a rocking speed Mass transfer - rocking speed

25 Comparison mass transfer CELL-culture shake flask STR (SUB) wave CELLtainer P/V (W/L) - 0, ,1 01 0,1 05 0,5 01 0,1 10 1,0 Gasflow (vvh) - < 1 0,5-1 < 1 k -1 l a (hr ) < < Microbial culture shake STR wave CELLflask (standard) (sparger) tainer P/V (W/L) < 0,5 2-3 Gasflow (vvm) - 1 < 01 0,1 05 0,5 k l a(hr -1 ) < 100 > > 300

26 Mixing times time (s) mixing wave 2L CELL-tainer 15L CT10-5L CT10-15L W10-2L 0 CELL-tainer 5L rocking speed (rpm) 25 Angle: 10 degrees

27 Batch and Fed-batch E.coli

28 Operational stability Growth of heterotrophic algea (TU Berlin) Runs > 1000 h Stable ph measurement No issues with bags Control of DO by shaking speed

29 CELL-tainer Comparison 100L shaker Shaker

30 Inoculation of a (large-scale) bioreactor? Step1 Step 2-n.Final process Many steps involved

31 Corynebacterium inoculation Experiment 1 Copy of shakeflask conditions Experiment 2 Controlled start

32 Result: simplification seed train Reduction of steps

33 Also suitable for cell-culture? YES! (Mab g/l) CELL-tainer wave (days) Productivity with PER.C6 fed-batch

34 CHO subclone 2 Bioceros Viable cell density x 10 6 Titer (g/l) Q values (pcd) Culture Time (days)

35 Fed-batch CHO CELL-tainer vs shaker

36 Scaleable?

37 Mass transfer large scale ( L)

38 Mixing times (75 150L)

39 Applicability CELL-tainer - technology

40 Questions of today - Answered Mixing and mass transfer in single use bioreactor: can we design a multipurpose single-use bioreactor? YES Can it be scaleable? YES

41 Acknowledgements Dr. Bout / Dr. Boon Bioceros Dr. Kiel / M. Andersen BioTest Aps Dr. G. Zijlstra DSM Biologics Dr. N. Chaudet c.s. Sanofi Pasteur Dr. Junne / F. Hillig TU Berlin A. Tromper / R. Engelhart CELLution Biotech Co-makers: CCM, DMT, Medistad, Dobit, etc.

42 new class single-use bioreactor designed to re-create ideal bioprocess conditions THANK YOU & QUESTIONS