Scouring the landscape of
the vast human proteome to identify biomarkers of
disease presents unique challenges for protein
researchers who rely on efficient, reproducible, and
high-throughput techniques for concentrating biological
samples, separating complex mixtures, and isolating
protein and peptide components. Ultrafiltration (UF) is
one commonly used method of concentrating solutions and
separating molecules in solution based on their size. UF
offers advantages over other techniques, such as
size-exclusion chromatography, including speed, ease of
use, and the potential for integration with automated,
high-throughput applications.
Millipore recently introduced an ultrafiltration
protocol designed for protein fractionation, a critical
step in proteomics research designed to reduce the
complexity of the proteome prior to protein
purification, separation, and analysis. Using Amicon
Ultra centrifugal devices, the protocol enables rapid
compartmentalization of small-scale protein samples.
"This protocol offers guidelines for researchers
to improve protein fractionation when using
ultrafiltration. Compared with existing techniques and
methodologies, this approach simplifies the process to
fractionate proteins based on size," says Jason
Whalley, product manager in the Bioscience division of
Millipore.
For high-throughput protein concentration, Sartorius
Vivascience offers a reusable 24-well ultrafiltration
frame — with the same dimensions as standard
microtiter plates for integration with robotic
liquid-handling instruments — that can accommodate 24
individual Vivaspin 500 ultrafiltration devices.
Simultaneous processing of 24 samples enables 30 times
concentration of 500-µL samples in 15 minutes,
according to the company. Each Vivaspin UF device has a
vertical membrane design and a built-in dead stop pocket
to prevent concentration to dryness.
Process Optimization and
Validation
To meet the need for low-volume process design,
optimization, and scale-up for small-scale protein
production in the research and clinical testing phases
of drug discovery, Pall has developed two new Tangential
Flow Filtration (TFF) systems, the Minim II and the
Centramate 500. Both are compact, bench-top systems with
integrated data-logging capability to facilitate process
validation.
Advantages of the Centramate include its sanitary
design. It is self-venting and completely drainable.
Because it fulfills the requirements for cGMP operation,
it can be used both for drug development and for
scale-down applications in drug manufacturing. The
system’s low working and holdup volumes allow it to
process small sample volumes — less than 200-mL
working volumes, according to Thomas Scholz, marketing
director for TFF Technologies at Pall. Pressure
transducers that monitor inlets and outlets and a flow
meter on the retentate side enable automatic flow
measurements and recording of data. A complete
documentation package is included with the system.
The Centramate provides an effective membrane area of
200 cm2 to 1 m2. The smaller Minim II, due on the market
in February 2007, has a membrane area range of 50 cm2 to
400 cm2 and can handle working volumes of 100-200 mL. It
also features a sanitary design in compliance with GMP
standards.
The Minim II can operate at pressures typically
associated with large-scale TFF processes, explains
Scholz. A touchscreen interface depicts the flow path,
documenting pump speed, temperature, and pressure
measurements directly on the flow diagram. The system
can accommodate up to three Minimate encapsulated TFF
membrane cassettes, each with a 50-cm2 membrane area.
The membrane cassettes can have the same or different
molecular weight cut-offs, allowing for flexibility in
process development and optimization applications.
UF Membrane Selection Guide
For help in selecting a filtration product for use in
early phase development, GE Healthcare recently released
a hardcopy version of its new UF/MF cross flow selection
guide and will follow with a Web-enabled version of the
guide toward mid-2007. The guide includes the company’s
ÄKTAcrossflow fully automated UF system designed for
process development. Using filters ranging from 40 cm2
to 150 cm2, the system has a minimum working volume of
25 mL. It is adaptable for use with either cross flow
cassettes or hollow-fiber cartridges.
With the introduction of the Kvick Start cartridge,
GE completed its portfolio of UF cassettes. Low working
and holdup volumes make the Kvick Start cassettes useful
in applications where starting material is limited,
including product and process development and scale-up.
Scheduled for release in 2007 are a new hollow-fiber
membrane optimized for the vaccine market and a kit that
will enable the ÄKTAprime bench-top chromatography
systems to run ultra-filtration/diafiltration protocols,
giving users of that platform access to cross flow UF
capabilities.
