Chromatography Forum

I have a 2.5L column with Sepharose 4 FF that I need to sanitize with 1M NaOH. After sanitization (with a contact time of 60 min), samples of the final rinsing water are taken to TOC and Bioburden analysis. The sanitization and sampling are performed in a Clean Room class C and to avoid external contaminations I also sanitize the external of the inlet and outlet tubing.
The question is that even with a low TOC, the acceptance criteria for bioburden (1 cfu per 10 mL) of the column sample is always out of specification.

Does anyone know what I might be doing wrong? Any sugestion?

Any comment would be helpful.

Thank you in advance.

Could you give some more detail about your sanitization procedure (Flow rate, volume, etc) and what type of samples are processed on the column? I've had experience with cleaning sepharose and other agarose/dextran based columns, but never measured TOC or bioburden after cleaning. I do recall that we were able to detect (but not quantify) proteins from previous samples remaining on the media even after a wash with 0.5 M NaOH. Our response was to increase the volume of NaOH used in the wash, thereby increasing contact time as well.