Vessel Sterility Testing and Operation

[tetonfl]

I am a recent chemical engineering graduate working in the biotech field. Having prior process engineering experience from an internship with a pharmaceutical manufacturing project, and seeing the level of understanding those chemical engineers had for sterile process design, I figured this would be a good spot to ask this question.


I am considering different methods of ensuring proper cleaning and contaminant free operating conditions for a continuous fermentation with a yeast culture. The only concern is biological pathogen contamination. There are no issues with having particulates or other debris in the final product.

There is a 200L vessel which is cleaned by DI water washes, base wash, acid wash, and steam. The vessel operates for 5 days before contaminants are visible under a microscope. We are working with other options, such as antibiotics and pH swings, to maintain operation for another 3 or 4 days before the contamination gets out of control and we have to shutdown.

I have read some books that suggest contamination is a fact of life in continuous fermentation processes and that attempting to maximize run time is more important than figuring out where the contamination came from. Is that true?

Are there any good books or resources on maintaining or testing process sterility? Sterility isn’t really the right word as we don’t want to kill the yeast, just the other organisms.

I know a riboflavin test would show surface wetting; however, is it even useful in this instance? The final steam cleaning should destroy any colonies the acid and base washes couldn’t get. Does the riboflavin degrade at a certain temperature or is there a similar marker with a thermal response/indicator that can be used to test for trouble spots in a steam cleaning?

A swab test could be used; however, the vessel is extremely difficult to disassemble and is housed in an open environment (large warehouse with open forklift doors). It would be difficult to validate that any contamination didn’t occur by just opening the vessel to swab it.

I have heard you can do a “yogurt” test and then the swab test. If you come back with any cells from the yogurt culture that would validate the swab test. Or we could just look for our yeast but we haven’t found any by doing so thus far.


If anyone has any experience with this sort of thing and could offer suggestions or point me in the right direction it would be greatly appreciate.

-Tom

 

[quark]

Riboflavin test is used to check the cleanability (i.e cleaning process effectiveness). By this you can identify and rectify dark spots. There is nothing you can do once the batch gets contaminated. You should be careful to see whether the vessel becomes sterile or not after sterilization. You can perform two kinds of tests.

One is keeping challange bacterium (micobacterium Stearothermophilus), at the darkest corner (probably the vent line nozzle) and check for Sterility Assurance Level. This also helps you in deciding your sterilization cycle duration.

Second and more assured method is to fill the vessel with sterile SCDM till the batch duration and check for contamination. Taking samples at regular intervals can tell you the story of contamination.

This is a pretty big subject and any basic microbiology text book can give you the details you required. Visit

http://www.fedegari.com

for sterility related knowledge. Come with specific questions, if any.