2008 Berge ACS Presentation

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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

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    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

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    .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

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    .00

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    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

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    Cerelose

    Fructose

    Maltose

    Sucrose

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    Comparison on Insoluble Fraction

    Percentage Insoluble

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    Insoluble% Cerelose

    Fructose

    Maltose

    Sucrose

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    Glycerol Runs

    Growth Comparison

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    .000 .000 .0000 .0000 .0000 .0000 .0000 .0000

    Glucose Glycerine

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    Titer Comparison

    Insoluble Titer

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    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