RESULTS AND DISCUSSION
TOC Measurement System Performance
According to the European good manufacturing practice (GMP) regulations, analytical methods used in biomanufacturing equipment
cleaning should be able to detect the established acceptable level of the residues or contaminant.14 Also, according to US FDA cGMP regulations and the USP, the suitability of all compendial testing methods used must be verified under normal laboratory conditions.15–16
Per the above regulations and considering that this is a quantitative test for impurities, the analytical parameters including
accuracy, precision, linearity, detection limits, and quantiation limits, were evaluated with their corresponding assumption,
per ICH Q2 (R1).9
The results obtained for each of the above parameters during the TOC analysis are summarized in Table 1. In the case of precision,
1.27% of RSD corresponds to 7.21 ppb of TOC. The linearity showed a fit correlation between ppb of TOC and the conductivity
signal from its full oxidation. The lowest amount of TOC quantified in 21 ppb (21 ng/mL) demonstrated high sensitivity of
this measurement system.
Table 1. Validated analytical parameters and the key results obtained
In addition, the suitability test was performed according to the procedure and specifications described in section 643 of
the USP31–NF26 and 2.2.44 of the European Pharmacopeia (EP, data not shown).16–17
Correlation Between TOC and Microorganisms
The bacterial and yeast cells commonly used in fermentation represent the "worst case scenario" in manufacturing system cleaning,
because they lead to organic contaminants such as nucleic acids, lipids, carbohydrates, proteins, and endotoxins. The TOC
test should provide a linear correlation between the measured molecules and the TOC response.
To analyze the relationship between cell concentration and TOC response, a correlation analysis was performed (Figure 1).
In both cases, correlation coefficients were >0.983, showing linear relationship between wet cell concentration and the TOC
values. Therefore, contamination with cells or any of those components can be quantitatively determined using the TOC measurement
in a wide dilution range. These data can be used to extrapolate the potential wet cell concentration with its corresponding
cell number estimate or relative organic carbon contribution in the final results of any cleaning process of a bioprocess
Considering that both bacterial and yeast cultures were harvested at an exponential growth phase, we assumed that the majority
of the cells were viable, although some lysed cells were not discarded.
It is significant to note that in first 27 ppb (27 ng/mL) of TOC, one could have a wet bacterial cell concentration of 1.29
mg/mL, which is equivalent to 106
E. coli cells. Similarly, in as little as 16 ppb (16 ng/mL) of TOC, one could have a wet yeast cell concentration of 0.826 mg/mL,
which is equivalent to around 103 yeast cells.
However, according to the WFI specification (500 ppb of TOC) indicated in EP16 and the USP17, the TOC values (27 and 16 ppb) are more than one order below the specification. Thus, the amount of residue found in a final
wash sample from manufacturing equipment cleaning could be considered as the "acceptable level" of undesirable residues. To
use this value as acceptable level, the TOC value of the water used for cleaning the manufacturing system must be taken as
the cleanliness reference.