NAMS 2002 Workshop - ICOM 2008

Biomedical and Biotechnology II – 2
Wednesday July 16, 10:15 AM-10:45 AM, Honolulu/Kahuku
Integrated Membrane-Based Sample Prep Approach for Viral and Microbe
Capture, Lysis, and Nucleic Acid Purification From Complex Samples
R. Baggio (Speaker), Millipore Corporation, Bedford, Massachusetts, USA -
rick_baggio@millipore.com
K. Souza, Millipore Corporation, Bedford, Massachusetts, USA
J. Murrell, Millipore Corporation, Bedford, Massachusetts, USA
L. Mullin, Millipore Corporation, Bedford, Massachusetts, USA
M. Aysola, Millipore Corporation, Bedford, Massachusetts, USA
J. Lindsay, Millipore Corporation, Bedford, Massachusetts, USA
G. Gagne, Millipore Corporation, Bedford, Massachusetts, USA
C. Martin, Millipore Corporation, Bedford, Massachusetts, USA
The detection of microbial and viral contamination in a timely, simple, and
effective manner is a concern of high interest to bioprocess workflows. Anaerobic
bacterial and mycoplasma detection based on growth and viral detection based
on infectivity assays are notoriously slow, labor intensive, and costly. Our
laboratory has developed an integrated membrane-based approach coupled to
quantitative PCR (qPCR) for the detection of Pseudomonas aeruginosa,
Propionobacter acnes, Mycoplasma hyorhinis,and Minute Virus of Mouse (MVM)
in both simple and Chinese Hamster Ovary (CHO) cell loaded samples at levels
as low as 100 CFU. In the sample preparation described in this work all cells in
the sample are captured and processed. By configuring prefilters, retentive
membranes, and affinity membranes in single or stacked interlocking devices
more aseptic processing could also be realized. The closed devices have been
engineered to be capable of multiple process work involving multiple and
discontinuous solution handling. By configuring size exclusion membranes and
affinity-based retentive membranes in sequential order, multiple process steps
are simultaneously carried out. The approach simplifies sample preparation steps
by combining size excluision chromatography and affinity chromatogarphy in a
unified membrane-based format. The separate steps of microbe capture, microbe
lysis, and nucleic acid purification are all performed in the devices, providing a
simplified method of preparation for challenging samples. These results
demonstrate the utility of an integrated method for sample preparation from
samples that mimic the complex bioreactor environment.