Post on 07-Apr-2018
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Inputs Outputs
Bacteria Metabolism
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Carbon Choices
Glucose (Cerelose)
Glycerol
Fructose
Sucrose Maltose
Lactose
Galactose Starches (after treatment with amylase)
Corn syrup
Molasses
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Glucose is Very Common Carbon Source
Glucose Pros
Was Cheap
Bacteria prefers it/Fast Growth
Easy measurement/On-line feed control possible Sold as powder
Cons
Low concentration to keep from crystallizing (~55%)
Acetate formation if overfed
- Batch level limitation (peak acetate formation)
Caramelizes with extended heat exposure
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Glucose Prices are Increasing Due to Corn Demands
Bio-fuels
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Why Investigate Carbon Sources?
If there was a glucose shortage, future processes mayuse back-up materials
Switching carbon sources may reveal areas ofimprovement for the fermentation process
Identify possible yield improvements for currentprocess
Further characterize the strain that we use in the
fermentation
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Study Objectives
Confirm which carbon sources will work forfermentation scheme
Determine growth differentials from varied carbonsource use
Determine production differentials from varied carbonsource use
Rank back-up carbon sources for process use
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Study System
E. coli fermentation producing therapeutic protein asinclusion bodies
12-L SIP fed batch fermentations
Batch media sterilized in two portions (nitrogen andcarbon separate)
Separate sugar and nitrogen feeds
2 sided pH control
Chemical Induction
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Sugar Batch Process Comparison
0
0
00
00
00
00
00
0 0 0 0 0 00 00 00
Cerelose
Sucrose
Fructose
Maltose
Galactose
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Fed Batch Comparison Runs
Galactose not used due to cost
Each sugar used three times
All runs verified to be carbon limited
Equivalent carbon basis in batch and feeds Feed and induction initiations based on same criteria
for each system
All runs progressed to same terminus (hours post
induction) All other run parameters identical
All samples processed at the same time
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Growth Comparison of Fed Batch Runs
Growth Comparison
0
00
000
000
000
000
000
.000 .000 .0000 .0000 .0000 .0000 .0000 .0000 .0000
Cerelose Fructose Maltose Sucrose
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Acetate Levels for Fed Batch Runs
Acetate Comparison
0
.00
0
.00
0
.00
0
0 0 00 00 00 00 00 00 00
Cerelose Fructose Maltose Sucrose
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Acid Consumption for Fed Batch Runs
Acid Comparison
.000
.0000
.00000
.00000
.00000
.00000
.00000
.000 .000 .0000 .0000 .0000 .0000 .0000 .0000 .0000
Cerelose Fructose Maltose Sucrose
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Sucrose Behavior
The high demand for acid points to a possible severecarbon limitation
Higher feed rates to be investigated
Possible advantage of using sucrose throughout run islost if high feed rates are needed, although there arework-arounds
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Titer Comparison of Fed Batch Runs
Insoluble Titer
0
0
0
0
0
00
00
00
Cerelose
Fructose
Maltose
Sucrose
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Comparison on Insoluble Fraction
Percentage Insoluble
00
00
00
00
00
00
Insoluble% Cerelose
Fructose
Maltose
Sucrose
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Glycerol Runs
Growth Comparison
0
00
000
000
000
000
000
.000 .000 .0000 .0000 .0000 .0000 .0000 .0000
Glucose Glycerine
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Titer Comparison
Insoluble Titer
0
0
0
0
0
00
00
00
Cerelose
Glycerine
Cerelose: 72.9% INS Glycerol: 72.6% INS
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Leveraging the Glycerol Advantage
Glycerol is up to ~80wt% soluble in water whilemaintaining similar viscosity (compared to 55%Glucose)
Overfeeds with glycerol do not lead to acetate
formation
High batch glycerol levels can be used to increase celldensity without feeding
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Conclusions
All carbon sources investigated will allow for cellgrowth and therapeutic protein production
Glucose or Glycerine proved to be best carbon choicein direct replacement scenario relative to growth
Glucose/Glycerine, Fructose, Maltose, Sucrose
Glucose, Glycerine, and Maltose had commensuratetiter levels Glycerine/Maltose/Glucose, Fructose, Sucrose
The two disaccharides used in the study had higherlevels of insoluble protein than monosaccharides andglycerine
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Special Thanks
Jerry Yang
Ken Lawson
Kiko Montes
Zoltan Baly
Min Choi
Mike Bolish
Shane Santiago
James Wing Mike Pritchard
Belle Puno