Advertorial
Retention of Mycoplasma at Elevated Pressure
and Cell Concentration by Pall Fluorodyne® EX
Grade EDT Filters
Notably, there is no standard test method or performance
criterion for characterizing a 0.1 µm rating for a given filter.
Mycoplasma retention performance will depend on how the
mycoplasma challenge testing was conducted. Therefore, to
ensure robust evaluation of filter membrane performance,
scientists at Pall Life Sciences have performed a mycoplasma
challenge study of Pall’s 0.1 µm rated filter membrane Pall
Fluorodyne® EX grade EDT, under high differential pressure
and an elevated mycoplasma challenge concentration under
conditions previously established to be highly penetrative.1
These highly penetrative Acholeplasma laidlawii mycoplasma
cells were used in a challenge of 0.1 µm rated Pall Fluorodyne EX
grade EDT filters at elevated differential pressures - 2 and 3 bard
(30 and 45 psid). Nine of these 47 mm diameter filter discs
(13.8 cm2) were each challenged with >1 x 107 colony forming
units (CFU)/cm2 of Acholeplasma laidlawii (ATCC 23260) in
1 liter of Mycoplasma Broth Base at 30 and 45 psid. An
additional set of nine filter discs was challenged at 3 bard
(45 psid) with a challenge exceeding 1 x 108 CFU/cm2. This
challenge level is ten times the minimum bacterial challenge
level recommended in the ASTM International Standard F838-05
for bacterial challenge of 0.2 µm rated “sterilizing grade” filters.
At the >1 x 107 CFU/cm2 challenge level and 2 barg (30 psig)
pressure differential, no penetration was observed, whereas at
3 barg (45 psig) differential pressure six of the nine samples
tested each showed penetration of a single CFU. At the higher
challenge level of > 1 x 108 CFU/cm2 also at 3 bard (45 psid),
there was no penetration of five filters, while the remaining four
filters (total of nine) showed penetration of only two to seven
CFU. This resulted in a minimum titer reduction of 108 (log
reduction value, LRV >8) in all cases, even under these
challenging high pressure, high mycoplasma challenge load
conditions. Refer to Figure 1 for a summary.
An examination of the results in Figure 1 shows that the risk of
penetration increases with increasing pressure (differential) and
with increased bacterial load. Consequently, the use of a lower
process pressure (2 bard [30 psid] or less) and presence of a
lower bioburden in the process fluid should significantly reduce
the likelihood of penetration by A. laidlawii through the 0.1 µm
Figure 1
1.00E+10
1.00E+09
10
Total Challenge
Average Recovery (N=9)
9
1.00E+08
8
1.00E+07
7
1.00E+06
6
1.00E+05
5
1.00E+04
4
1.00E+03
3
1.00E+02
2
1.00E+01
1
1.00E+00
0
30 psid @ >
1x107 CFU/cm2
Average Total Recovery (CFU/Filter)
Mycoplasma are diminutive bacteria without a rigid cell wall
that range in shape and size (< 0.2 to ≥ 0.5 µm). Due to
their presence in water, soil, plant, animal and human origins,
mycoplasma have an unfortunate propensity for contaminating
mammalian cell lines and cell culture media. Contamination
results in decreased viability and cell counts during cell culture,
as well as reduced activity and product yield. The unusually
small size and lack of a rigid cell wall in mycoplasma make
them particularly adept at penetrating traditional 0.2 µm rated
sterilizing grade filters, to varying degrees. As a result, protection
from mycoplasma contamination by filtration requires the use of
0.1 µm rated filters.
filter membrane. The results in Figure 1 also demonstrate the
high degree of sterilization assurance capability achievable with
Pall Fluorodyne EX grade EDT filters despite aggressive
challenge conditions in all cases.
Total Challenge (CFU/Filter)
Michael Moussourakis, Marketing Manager
45 psid @ >
45 psid @ >
1x107 CFU/cm2 1x108 CFU/cm2
Effect of pressure and mycoplasma challenge load on penetration of A.
laidlawii mycoplasma through Pall Fluorodyne EX grade EDT filter membranes
(Part Number EDT04725). Data shows no penetration of the filter at a
challenge pressure differential of 2 bard (30 psid) (challenge level ≥107
CFU/cm², while increasing pressure differential to 3 bard (45 psid) and
increased mycoplasma load to > 1x108 CFU/cm² allowed a low level of
penetration. Note: The validated mycoplasma retention claim for Pall
Fluorodyne EX grade EDT filter cartridges and capsules is > 10 LRV for both
A. laidlawii and M. orale under standard laboratory challenge conditions at
2 bard (30 psid).
Fluorodyne EX EDT filter cartridges feature a highly permeable
0.1 µm rated membrane assembled in a patented high area
10-inch laid-over pleat and narrow core construction filter
module with exceptionally high filtration area (0.95 m2 EFA per
10” module). The cartridge provides enhanced capacity for
sterilizing filtration of biological fluids (e.g. culture media) and
mycoplasma sterility assurance under even the most stringent
testing conditions. To reduce the risk of mycoplasma
contamination in cell cultures and aseptic fill validation media,
0.1 µm high mycoplasma retention rated Pall Fluorodyne EX
grade EDT filter cartridges and capsules should be considered
a lead candidate.
Please see Folmsbee and Moussourakis 2 for further details of
this study. Pall is pleased to work with you to qualify the most
appropriate filter to ensure sterilization of your drug or vaccine
product. Please contact Michael Moussourakis at
mmoussourakis@pall.com for further information.
Pall Fluorodyne EX grade EDT filters. Unrivalled
mycoplasma control with superior value.
References
1. Folmsbee, M.; Howard, G.; McAlister, M., Nutritional effects of culture media
on mycoplasma cell size and removal by filtration. Biologicals, 2010, 38, (2),
214-217.
2. Folmsbee, M.; Moussourakis, M., Retention of Highly Penetrative Mycoplasma cells (Acholeplasma laidlawii) by A New 0.1 µm Rated Membrane
Filter at Elevated Pressure and at an Elevated Challenge Concentration.
BioProcess International, 2012, 10(5).
w: www.pall.com/biopharm
e: biopharm@pall.com
© 2012 Pall Corporation. Pall,
, and Fluorodyne are trademarks of Pall
Corporation. ® indicates a trademark registered in the USA. GN12.5029
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