Techno-economic analysis of semicontinuous production of - - PDF document

▶
techno economic analysis of semicontinuous production of
SMART_READER_LITE
LIVE PREVIEW

Techno-economic analysis of semicontinuous production of - - PDF document

Feb 19, 2023 91 likes •181 views

4/4/2018 1 Techno-economic analysis of semicontinuous production of recombinant butyrylcholinesterase in transgenic rice cell suspension cultures Jasmine M. Corbin 1 , Matthew J. McNulty 1 , Karen A. McDonald 1,2 , and Somen Nandi 1,2 . 1.

slide-1
SLIDE 1

4/4/2018 1

Techno-economic analysis of semicontinuous production of recombinant butyrylcholinesterase in transgenic rice cell suspension cultures

Jasmine M. Corbin1, Matthew J. McNulty1, Karen A. McDonald1,2, and Somen Nandi1,2.

  • 1. Department of Chemical Engineering, University of California, Davis, CA, USA
  • 2. Global HealthShare Initiative, University of California, Davis, CA, USA

1

March 21, 2018 ACS 255th National Meeting: Disruptive Bioprocessing & Process Integration

Plant Cell Suspension Cultures

2

 Problem: Need for reliable, safe, and scalable methods for

production of biologics

 Approach: Plant cell suspension cultures

Advantages:

  • Inexpensive
  • Intrinsically biosafe
  • Complex products

(post-translational modifications)

Limitations:

  • Immune response to

plant glycans

  • Low growth rate and

protein expression

  • Not well characterized
  • r developed
slide-2
SLIDE 2

4/4/2018 2

Butyrylcholinesterase from Transgenic Rice Cell Suspension Cultures

3

Rice cell suspension culture:

 Dedifferentiated cells from Oryza sativa  Codon optimized human BChE gene  Inducible promoter activated by sugar starvation Pan YM, et al. 2009. J Phys Chem.

Butyrylcholinesterase (BChE):

 Bioscavenger for nerve agents  Human BChE from plasma = $20k per 400 mg dose  340 kDa tetramer  14 disulfide bonds  36 N-glycosylation sites

Side view Top view

Semicontinuous Bioreactor Operation

4

  • 1. Expression level:

~60 µg BChE/g FW (4 mg/L culture) in 10L bioreactor

  • 2. Continuous 5/5 day

cycling for >6 months with no decrease in expression level

Corbin JM, et al. 2016. Frontiers in Plant Science. 7:1-9.

slide-3
SLIDE 3

4/4/2018 3

Laboratory Scale Downstream Processing

5

Microfiltration Homogenization AEX Chromatography (DEAE) Affinity Chromatography (Hupresin) Concentration & Diafiltration (TFF) Clarification

SDS-PAGE Western blot

>95% purity of active BChE

Corbin JM, et al. 2018. Biotechnology and Bioengineering:1-10.

Design Parameters and Assumptions: Upstream

6

Current (Laboratory Scale) Projected (Manufacturing Scale)

Initial culture density: 7 10 g FW/L Final culture density: 70 100 g FW/L Doubling time: 4 3 days Time, Initial to final density: 13 10 days Growth duration: 5 4 days Expression duration: 5 3 days Total cycle time: 10 7 days Expression level: 60 200 mg BChE/kg FW 4 20 mg BChE/L culture

Production Target: 25 kg pure BChE year

slide-4
SLIDE 4

4/4/2018 4

Design Parameters and Assumptions: Downstream

7

Step Current (Lab Scale, Experimental*) Projected (Manufacturing Scale) Step Recovery Process Recovery Step Recovery Process Recovery Crude Extract 100% 100% 100% 100% Clarification 100% 100% 96% 96% UF/DF (TFF) 95% 95% 97% 93% IEX (DEAE) 75% 71% 80% 73% Affinity (Hupresin) 60% 43% 85% 62% UF/DF (TFF) 95% 41% 95% 59%

*Corbin JM, et al. 2018. Biotechnology and Bioengineering:1-10.

Overall Downstream BChE Recovery: 60%

Upstream: Seed Train

8

Each seed train step:

  • Inoculate at 10 g/L
  • Grows to 100 g/L over

10 days (τD = 3 days)

  • Used to inoculate next

step at 10 g/L

slide-5
SLIDE 5

4/4/2018 5

Upstream: Production Reactor

9

4x 20kL production reactors

  • Grow 4 days (20kL)
  • Medium exchange (20kL)
  • Express 3 days (20kL)
  • Harvest (10kL)
  • Medium exchange (20kL)
  • Repeat for 6 months before

shutdown, CIP, SIP

Downstream: Clarification and Filtration

10

  • Extract at 1:3 ratio

(biomass to buffer)

  • Concentrate 10x
  • Diafilter 4x
slide-6
SLIDE 6

4/4/2018 6

Downstream: Chromatography and Formulation

11

  • All chromatography bind and elute
  • DEAE recovery = 80%
  • Hupresin recovery = 85%

Final product:

  • 59% Recovery
  • 95% Purity

Economics: CAPEX, OPEX, COGS

12

Parameter Unit Section Base Case

CAPEX $ million Upstream 141.6 Downstream 24.4 Total 166.0 OPEX $ million/ year Upstream 2.6 Downstream 32.0 Total 34.6 COGS $/g BChE Upstream 115 Downstream 1,432 Total 1,547

$620 per 400 mg dose

Raw Materials 69% Labor-Dependent 17% Laboratory/QC/QA 6% Consumables 8%

Annual Operating Cost Breakdown

slide-7
SLIDE 7

4/4/2018 7

Conclusions and Future Work

 Developed base case model for

manufacturing scale production

  • f recombinant BChE in

transgenic rice cell suspension culture

 Developed tool for future process

design and analysis

 Refine costs, process times,

process parameters

 Minimize equipment down

time

 Alternate cases  Reactor scheduling  Clarification equipment  Sensitivity analysis  Expression level  Affinity resin binding capacity,

recovery

13

BChE Source Cost per 400 mg Dose Human plasma $20,000 Rice cell culture $620 >30-fold reduction in cost

Corbin JM, et al. 2016. Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures. Frontiers in Plant Science. 7:1–9.

Corbin JM, et al. 2018. Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures. Biotechnology and Bioengineering:1–10.

Tusé D, et al. 2014. Manufacturing economics of plant-made biologics: Case studies in therapeutic and industrial enzymes. Biomed Res. Int. 2014.

Nandi S, et al. 2016. Techno-economic analysis of a transient plant-based platform for monoclonal antibody production. MAbs 8:0.

14

Acknowledgements References

http://mcdonald-nandi.ech.ucdavis.edu/