Validating Sterile Filtration: Overcome the Fear of Failure 2

From Tolliver and Schroeder (1983) courtesy of Microcontamination
B. diminuta should not be viewed as a universal model organism, as some native bioburden may be a better alternative as challenge organism, being close to the actual process settings. Unfortunately, rare penetrations of sterilizing grade filters have caused an exaggerated doubt in the reliability of filtration. Appropriate process validation though should render such doubts and be trusted by even the most critical reviewer of sterile filtration..
Sources of Variability: Size and Shape
B. diminuta varies in size and shape, depending on how it is cultivated. Back in 1967, Bowman and colleagues described the B. diminuta size as 0.3 × 1.0 μm [7]. However, in 1978, Leahy and Sullivan found [8] that the organism grown at 30 °C and incubated for 24 hours in saline lactose broth, a minimally nutritional medium for that microbe, yielded cocci-like cells approximately 0.3 × 1.0 μm (Figure 1). Similar considerations have to be accounted for when challenge tests are performed with native bioburden forms.
B. diminuta are typically cultivated to develop as spherical a form as possible, since spheres are least amenable to retention. Thus, Leahy and Sullivan proposed, back in 1978, that it be used as the model organism for 0.2/0.22-μm-rated membranes, partly because of its size relative to the 0.2-μm dimension [8].
Subsequently, the FDA designated it for that very purpose [9], defining a sterilizing filter as one that retains a minimum of 1 x 107 cfu of Brevundimonas diminuta ATCC 19146 / per cm2 of effective filtration area (EFA).
Although it isn’t the smallest organism known, B. diminuta was considered diminutive enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. The smaller the test organism, goes the logic, the more likely that its removal by a filter would assure the sieve retention of larger organisms.
However, ease and safety of cultivation and handling are also important considerations. In 2001, Sundaram and colleagues found an increasing number of cases where filtration in 0.2/0.22-µm-rated membranes failed to yield sterile effluent [10]. Experimental studies showed that penetrating organisms had shrunken because they had been cultivated in broths that were nutritionally inadequate. In such cases, the physicochemistry of the suspending fluid may serve to alter the size of the suspended organisms as expressed by the Donnan equilibrium consequent to ionic strengths.Leahy Organism Shape
Organism Shrinkage During Processing
Sundaram’s team [11] also found that organisms underwent size changes after exposure to certain drugs. In cases with 0.2-µm-rated membranes, the researchers found, the larger pore size could only provide sterile effluent and/or a high titer reduction with regard to certain organisms for various lengths of time, before penetration occurred.
Penetration times varied from 24 to 96 hours, and the cumulative challenge at which penetration was first observed ranged from 1.2 x 107 to 1.1 x 108 cfu/cm2. Two 0.2-µm rated Nylon-66 filters in series were unable to fully retain Ralstonia pickettii (now Burkholderia pickettii) with penetration observed at 72 hours, corresponding to a cumulative challenge of 2.4 x 107 cfu/cm2. The more extensive penetration of the Nylon-66 membranes, compared with the PVDFs, is in keeping with their greater degree of openness, as Krygier and colleagues showed in 1986 [12].
As a result, it has been suggested that 0.1-µm-rated membranes be substituted for their 0.2-µm-rated counterparts.
Sundaram’s team evaluated five 0.1-µm-rated membranes and found that they yielded sterile effluent over the entire duration of the test (120-196 hours), up to challenge levels of 5.7 x 107 to 2.0 x 108 cfu/cm2. Similar results were obtained with the PVDF filters tested; no B. pickettii were detected at challenge levels of 5.9 x 107-6.0 x 108 cfu/cm2.
In addition, all 0.1-µm-rated filters tested provided consistent and complete retention of B. pickettii for the entire duration of the test (120-192 hours), suggesting that the smaller pores would ensure sterile product at conditions where penetration could occur through conventional 0.2- and 0.22-µm-rated sterilizing grade filters. Proponents argue that using 0.1-μm-rated membranes would permit longer term formulation and filtration operations. In fact, 0.1 µm-rated filters may be the best choice for long-term filtrations.
However, penetration has also been found in 0.1 µm-rated filters. In 1999, Sundaram’s team found that B. pickettii, when its size was so affected, could be retained by certain 0.1-µm-rated filters. But, in a similar situation, they found that only four of seven commercially available 0.1-μm-rated membranes could remove a particular organism. Just because one type of membrane so classified may provide proper retention, does not mean that any other 0.1-µm-rated membrane can also be depended upon for a like result.
It is important to remember that today, there are no industry standards by which 0.1 filters can be judged. In addition, more research clearly needs to be done into the kinetics behind the organism’s size changes, evaluating different organisms in different fluids.

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