Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification.
OBJECTIVE
Contamination by Mollicutes is a significant challenge for research laboratories and biopharmaceutical industry. It leads to alteration of results or production quality as well as loss of time, materials and revenue. These organisms can czoriginate from mammalian, avian, insect, plant or fish cells. Culture-based methods may require 28 days to detect Mollicutes. Traditional microbiology could advantageously be replaced by nucleic acid testing for earlier detection.


RESULTS
A membrane filtration-based concentration of the Mollicutes has been coupled to real-time transcription-mediated amplification (real-time TMA) to demonstrate these advantages. The eight species required by European Pharmacopoeia have been tested and were detected with sensitivity below 100 CFU per 20-ml sample. Co-culture experiments, in which Mollicutes are grown with CHO-S (suspension) or HEK 293 (adherent) cells, were also performed to respectively mimic a bioreactor or flask contamination. Despite the fact that Mollicutes can attach to or invade mammalian cells, they were consistently detected over multiple days.


CONCLUSIONS
the sample preparation and amplification method used in this study increases sensitivity and reduces time-to-result for detection of Mollicutes.


CONCLUSIONS
the described system allows real-time monitoring for microbial contamination of cell-based processes and products for the biopharmaceutical industry.
Laborde
S
S
Millipore, Bioprocess division, Process Monitoring Tools, European Development & Industrialization, Applied Biology Department, 39 route industrielle de la Hardt, 67120 Molsheim, France. sandra_laborde@millipore.com
Degrave
A
A
Lehmann
D
D
Jouette
S
S
Rofel
C
C
Muller
T
T
Hertzog
N
N
Rook
M
M
Ribault
S
S
D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D016466:CHO Cells; D002460:Cell Line; D002477:Cells / Q000382:microbiology; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D008973:Tenericutes / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic