Large-Scale Production of Recombinant Proteins Lecture 6 ISAT 451, Fall 2003 College of Integrated Science and Technology James Madison University

Large-Scale Protein Production Most commercially valuable proteins (industrial enzymes or therapeutic proteins) require living cells to make the product Although cells can be readily grown in a standard 200 ml laboratory flask, commercial production may require containers in excess of 10,000 liters What are the limitations of large-scale production?

Microbial Growth Conditions Sterile culture medium containing a carbon source and essential salts or an enriched medium Pure culture for inoculation oxygenation and mixing Regulation of temperature Regulation of pH

Microbial Cell Growth

Batch Fermentation Sterile medium is inoculated and fermentation proceeds without the addition of fresh growth medium.

Fed Batch Nutrients are added at intervals during fermentation. No growth medium is removed.

Continuous Fermentation Fresh growth medium is added continuously during fermentation and spent medium is removed. Fermentation can last up to 1,000 hours.

Why do Cells Stop Growing? Depletion of nutrients Lack of oxygen Change in pH Growth inhibition from metabolic end products

Stirred Tank Bioreactors An external motor is used to agitate the growth medium with impellers. Sterile air or combinations of gases can be introduced (sparging)

Bubble Column Bioreactor Introduction of sterile air or pure gases is used for agitation and oxygenation.

Internal-Loop Airlift Bioreactor Sterile air or pure gases are introduced to create circulation internal to the reactor.

External-Loop Airlift Bioreactor Sterile air or pure gases are introduced to drive circulation through an external loop.