While a reasonable approximation of conditions (cleaning agent type, cleaning concentration, temperature, and time) can be obtained from relatively simple laboratory studies for the “washing” step of a CIP cleaning procedure, simple lab studies are less useful for the rinsing conditions, and specifically for the rinsing time and/or rinsing volume. Why is this the case? The main reason is that the washing step is fundamentally an issue of time of contact with turbulent flow. For the rinsing conditions, it depends more on the geometry of the equipment and/or the presence of any hindrances to effective rinsing (such as dead legs). For that reason, the best way to select appropriate rinsing conditions is to utilize actual production equipment or suitable scale-up equipment.
These rinse studies should be done following a washing step that has been shown (either in lab studies or in cleaning processes for other products) to be reasonably effective provided that robust rinsing follows that washing step. In addition, we should realize that a series of smaller rinse volumes will be more effective in reducing residue levels as compared to one continuous rinse of the same total (cumulative) volume. That is, if we think we should use 1000 liters of rinse water, better results will be achieved by using five (5) distinct rinses each with 200 liters as compared to one continuous rinse of 1000 liters. It also helps to allow each rinse to drain as completely as practical before going to the next rinse. Also, it may be counterproductive to increase the number of rinses (and therefore decreasing the volume of each rinse) to such a number that the each separate rinse volume does not adequately contact all surfaces of the equipment.
So, in a rinsing study with multiple rinses the approach should be to measure at the various rinse intervals something (a “marker”) that is indicative of the completion of rinsing. Since most CIP rinsing involves initial recirculating rinses followed by a final once-through (non-recirculating) rinse, we’ll focus on that situation. Let’s assume for this example, we will perform four (4) 200 liters recirculating rinses followed by a single once though rinse of 200 liters. So after the completion of each of the first four recirculating rinses, we collect a sample of that rinse and measure the marker.
The marker should be something that I expect to decrease significantly with successive rinses. That marker and the associated analytical method could be based on a specific molecule (such as the API). It could also be based on a general measurement such as TOC (useful if there are significant organics present) or conductivity (useful if alkaline or acidic cleaning agents or used). My preference would be measurement of both the API along with either TOC or conductivity. [Note for biotech: I would probably use both TOC and conductivity, since any active proteins will be degraded.]
Measuring that marker can be done by selecting any portion of each recirculating rinse as it exits the equipment. While there may be minor variations between the first portion and the last portion of each individual rinse, if it is of concern (or just to provide evidence that it doesn’t make a difference), samples from the beginning and the end of the rinse could be taken as the rinse leaves the equipment.
Then, in the final (once-through) rinse, the situation is slightly different. For this rinse it is at least theoretically possible that the marker level in the first portion of that once-through rinse could be higher than in the last portion of that same final rinse. The reason this may happen is not unique to this final rinse, but is an expectation of any once-through rinse. While this difference is a possibility, it is not a logical requirement if the value of the both the initial and final portion of that once-through rinse all had reported values below the LOD of the marker. In that case, I still suspect the true value is lower in the final portion, but with my analytical method I can’t clearly establish it. So I would like to do one of two things. One option is to collect the entire amount of that once-through rinse and measure the marker in that “composite” sample to determine a marker value of the “entire” final rinse. A second option is collect samples of the once-through rinse at various intervals and average them (or perhaps integrate them based on the portions of the final rinse each sample might represent). In either option I would have a reasonable estimate of the value of the marker in that once-through rinse. So now it is time to analyze that data for the marker as a function of the rinse number. Here are several possible outcomes with all rinses being recirculating except for the last rinse.
Example 1 – with marker as the API itself with L4c of 15 and LOD of less than 5. Here are data for the various rinses. The “relative reduction” factor for rinse “n” is calculated as the marker value for rinse “n-1” divided by the marker value for rinse “n”.
| Rinse No. | 1 | 2 | 3 | 4 | 5 | 6 |
| Marker Value | 50000 | 5000 | 500 | 50 | 5 | <5 |
| Relative Reduction | N.A. | 10 | 10 | 10 | 10 | N.A. |
So, how may rinses should be selected? In this case five (5) rinses should be adequate to obtain acceptable rinse limits. However, if swab sampling for the API is also done, acceptable values for swabbing should be confirmed after five rinses. [Note that for this example, while in the study the fifth rinse was recirculating, I would probably convert that to a once-through rinse going forward; while not exactly a fully logical conclusion (the recirculating rinse might take four minutes, while the once-through rinse might last only one minute), it is probably a reasonable approach.]
Example 2 – with conductivity as the marker and LOD for conductivity of less than 0.2: Here are the data for the various rinses.
| Rinse No. | 1 | 2 | 3 | 4 | 5 | 6 |
| Marker Value | 30000 | 3000 | 300 | 30 | 3 | 0.3 |
| Relative Reduction | N.A. | 10 | 10 | 10 | 10 | 10 |
So, how may rinses should be selected? In this case the value for the marker continues to decline into the sixth rinse. Some may choose to continue for a total of six rinses. Others may choose only five rinses based on the data on the API in Example 1 provided that the acceptance limit for alkaline cleaning agent was being met by the marker value of 3 after the fifth rinse. As with Example 1, if swab sampling is to be done, acceptable values for swab sample should be confirmed before a final decision is made on the number of acceptable rinses.
Here are some additional comments that should be considered in selecting the number of rinses required using studies of this type.
Comment A: In the two examples given, the relative reduction is the same for each rinse. In some situations, the reduction factors may decrease in the later rinses, most likely due to the marker being “held up” in some way as rinsing processed. This can be due to higher levels of a “difficult to clean” marker being left on surfaces or to “dead legs” in the flow paths. The best remedy for this type of situation is to increase the robustness of the washing step (longer time, higher temperature, and/or higher cleaning agent concentration) or to eliminate unacceptable dead legs. In an ideal situation, the reduction factor should be the same as rinsing proceeds; but as I have taught many times, we don’t necessarily live in an ideal world.
Comment B: If it is unclear which marker should be evaluated, consider selecting multiple markers and basing any decision on the appropriate number of rinses after evaluating the actual data on successive rinses.
Comment C: If there are situations where it is needed to select a possible rinse time before cleaning of a specific product is to be done, it is certainly possible to clean equipment with no product (or a placebo product) in the equipment. In this situation a marker for the cleaning agent can be used (which may be possible unless cleaning is done with water alone), using conductivity or TOC as the analytical method. In this situation, performance with a product, perhaps with both rinse and swab sampling, is required for the API itself before the acceptability of the number of rinses is clearly established.
Comment D: This technique may also be used to evaluate any changes in the cleaning situation where an unexpected failure has occurred in order to investigate a possible root case.
Comment E: This technique is certainly not a requirement that must be done. If you have been using a cleaning procedure with an established rinsing protocol, and the routine monitoring and yearly confirmation protocols have been consistently acceptable, look for other ways to improve the cleaning validation program rather than doing rinse studies described here. That said, perhaps your company is doing excessive rinses, and significant resource savings may be possible by reducing the amount of rinse water or rinse solvent utilized.
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