But what does the specification and the reported value on a C of A mean to you and your experiments ? How do you know if this is important for your work ?
This Tech Blog is a brief discussion on Endotoxin in Fetal Bovine Serum and its relevance to your cell culture experiments.
What is endotoxin ?
Endotoxin is collective term for complex lipopolysaccharide (LPS) which is a major component of the outer membrane of most gram negative bacteria. Endotoxin Bacteria shed endotoxin in small amounts when they are actively growing and larger amounts when they are dead.
History of FBS Production
FBS is a biological product and during its processing there is potential for bacterial contamination and growth prior to final sterile filtration and filing into serum bottles. It is at this time that endotoxin can be shed from bacteria and contribute to the endotoxin level of the serum. Historically the degree to with steps were taken to control bacterial contamination and growth during FBS production was not as controlled as it is today. The level of endotoxin in raw sera could vary quite dramatically, but improved methods of blood collection resulted in reduced Endotoxin levels. Nevertheless, if raw sera is not processed with care, bacterial growth will result in higher Endotoxin levels. At the time serum companies established different Grades of FBS ,in part based on the level of Endotoxin in a batch of serum. The lower the level of endotoxin in a batch of FBS suggests that the sera was processed with more care and is generally considered “higher quality” for the purposes of cell culture.
Endotoxin Specifications were established to help differentiate between higher and lower quality of FBS batches. An Endotoxin Specification is the maximum concentration of Endotoxin permitted for a given “Grade” of FBS. Batches of FBS with an endoxin level that fall below the Specification can be included in the Grade; batches over the level are classed as another Grade or rejected altogether. The Specification is stated on the C of A for theLot and is a defining constant for the given FBS Grade.
For example, NorthBio’s Ultra-Low Endotoxin Grade of FBS has an Endotoxin Specification of < 0.05 EU/ml; while our Cell Culture Grade FBS an Endotoxin Specification of < 10 EU/ml.
What is more important to the cell culturist is the Endotoxin reported “Value”. The reported Value sits beside the Specification on the C of A. The reported Value is the actual data value of endotoxin for that specific FBS Lot, as determined by QC testing. This value will vary between different Lots of FBS, because different lots are produced from raw sera batches that have different inherent amounts of endotoxin.
Important Point: Although one might think that a higher Endotoxin Reported Value would indicate that the Lot is of lesser “quality” than anotherLotwith a lower value, this is not necessarily true. The difference between a reported value of 0.1 EU/ml and 5 EU/ml will make no difference to most cell culture work, (remember, the Specification is < 10 EU/ml). However, a value of 50 EU/ml might not be desirable for research cell culture use and the sera is better suited for Diagnostic applications (eg. Blocking or in vitro diluent). However, some industrial users prefer a high Endotoxin Level for their cell culture precisely because it stimulates Cell Proliferation and/or protein production, and this is the only performance effect that they want from the sera. In this case, the High Endotoxin Lot would be considered a very good Quality.
What is the importance of Endotoxin to Biotechnology and Cell Culture
BiopPharmaceutical and Vaccine Manufacturers have 2 concerns about endotoxin. The first is that Endotoxin is a pyrogen with unwanted effects on humans or animals. In this regard, the endotoxin concentration in a final, injectable drug or biological product must fall below safe, regulated levels (not discussed here). The second concern is the influence that endotoxin can have on cell based biological expression systems used to produce the vaccine/biopharmaceutical product. This concern is shared by scientists employing cell culture for their research.
The effect of endotoxin varies greatly for different cell types and cell lines. The effects range from membrane and morphological changes, effects on secretion and gene expression, powerful mitogenic effects, and synergistic effects that are due to indirect effects of endotoxin on cells.
Due to the varying influence of endotoxin on cell expression, a researcher should be aware of how endotoxin might effect the specific cell types they are culturing. Awareness of cell surface receptors on cells used can alert a researcher to the potential for endotoxin sensitivity.
For more additional reading about Endotoxin and cell culture, see The Significance of Endotoxin to Cell Culture and Biotechnology, (LAL Update, Vol 16 No. 1) by the Associates of Cape Cod Incorporated.