Filled units must be incubated as soon as possible after filling. Regulators, the FDA in particular, are anxious that all units are incubated (with the exception of those without caps, obvious cracks, etc.). This is intended to include those "perfect" units that may in practice never be released, e.g., units cleared off the line after a stoppage or similar event. Regulators prefer incubation for information purposes, with indirect rather than direct impact on the success or failure of the trial.
When media are added in the laboratory after a solid placebo is filled, it is critical that the interval between filling the placebo and adding the medium is as short as possible. This prevents contaminating microorganisms dying off in the placebo. The argument against this, premised on genuine product contaminants dying off within the seven or 14 days' sterility test quarantine before release, is not valid. The media fill is intended to disclose process contamination, not the probability of nonsterility in product within its marketed shelf life. The maximum interval between filling and media addition should be validated by inoculation of the placebo and then tracking recoverable survivors over time.
Incubation of media fills is almost universally done for 14 days. This probably originates in the pharmacopoeial sterility tests where none of the major pharmacopoeias have for many decades asked for any longer incubation period. The exception to this is the Australian regulatory agency (the Therapeutic Goods Agency, TGA) which asks for 21 days' incubation to comply with its sterility test. Studies by the TGA have been influential in driving the USP and PhEur sterility test incubation period for the membrane filtration method up from seven to 14 days, even though there is no clinical evidence that the seven-day test has failed to protect the public. If the media fill is to be considered as an exhaustive search for potential viable microbial contaminants then the duration of incubation is potentially limitless. It is well known that some coryneform bacteria require 28 days or more incubation to produce visible turbidity in TSB. It is probably good conservative advice to incubate validation media fills beyond the 14-day period and justify future routine media fill incubation at 14 days or whenever the last contaminant was detected in the extended validation exercise, whichever is the longer.
There has been some controversy over the temperature of incubation for media fills — 20-25°C or 30-35°C. Any choice will always be open to criticism. Both temperature ranges (and probably some others, too) can be reasonably justified. Incubation at both temperatures is widely used, but this still leaves the decision over which temperature should be used in the first seven days, and which in the last seven days of incubation (or indeed should there be another pattern?). Once again both options are justifiable, and neither is worth an acrimonious argument with a regulatory inspector. One can only hope that facilities subject to inspection by different national agencies do not encounter single-minded inspectors with differing outlooks.
It is usual to incubate the filled units for seven days in their normal orientation, and for seven days upside down. The principle is to ensure that all of the internal surfaces of the container and closure are bathed in media for long enough to allow any adherent contaminants to be resuscitated, recover and grow. Almost always incubation in the correct orientation takes place over the first seven days, and upside-down incubation in the second period of seven days. The opposite approach could be as well justified.
The amount of media filling each container should be sufficient to reach halfway up the height of the container so that every internal surface is bathed by the medium for at least seven days. This is not always done. This factor should be taken into account when determining how the media fill is conducted.
It is advantageous to know if there are any contaminants in the media fill as soon as possible. Visual inspection, without disturbing the units, is normal on a daily or every-second-day basis. A thorough visual inspection should be conducted at seven days when the units are inverted, and 14 days when incubation is complete. Damaged or cracked units may be excluded from the results. The total number of units checked at the end of incubation, plus any removed for reasons of damage, should reconcile exactly with the numbers filled and presented for incubation. Reconciliation limits such as plus or minus 5% used in other aspects of pharmaceutical manufacture are unacceptable.
Visual inspection should be undertaken in good daylight or artificial light by personnel with good eyesight. These personnel should be subject to periodic sight tests. Turbidity is the typical indication of microbiological growth, but personnel assigned to this task should also be alert to the possibility of pellicle formation on the surface of liquid media and other forms of microbial growth. Visual inspection becomes more difficult with tinted glass containers. It is certainly most awkward for ophthalmic ointments where the contents have to be squeezed out (usually on to white paper), and examined for growth as indicated by the red colouration produced from the oxidation of tetrazolium chloride, or by the presence of bubbles.
The microorganisms from every contaminated unit obtained in any media fill should be subcultured, purified and identified to species level. Where possible the tray number and time of filling of every contaminated unit should be retained. The identity of any microbial contaminants is a major part of the information content of the media fill. Where possible the identified microorganisms should be related to the events happening at the time when the contaminated unit was filled. This view appears to contradict the apparent obsessiveness of many pharmaceutical manufacturers, microbiologists, regulators and standards writers, to place the emphasis of contaminated media fills on the numbers of contaminated units, or on the proportion of contaminated to uncontaminated units. There is practically no information content in knowing that there were two contaminated units in a media fill of, say, 4000 units. Conversely, knowing that the two contaminants were, for example, pseudomonads or micrococci, points the experienced microbiologist to the most likely source of contamination and allows intelligent diagnosis of the problem and focused corrective or preventive actions.
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