Purify Proteins Fast with FPLC

Most biologists are familiar with high-pressure (or -performance) liquid chromatography (HPLC),
a chromatographic technique used to separate proteins, peptides, metabolites and more. They may not
be as well versed in fast protein (or performance) liquid chromatography (FPLC).

Used to separate and purify proteins, FPLC has much in common with HPLC, though FPLC generally
uses a higher flow rate and lower pressures to move the buffers and sample through the separating
column.

In many cases, molecules may be separated using either method, but there are differences. Here are
some tips from FPLC experts on when to use it.

HPLC or FPLC?
Traditionally, FPLC and HPLC were differentiated by pressure. Today many agree that the best criterion
is what the researcher plans to do with the product. Essentially, the question boils down to whether your
separation work is preparative or analytical, says Pia Liljedahl, global product manager at GE Healthcare
Life Sciences. Although HPLC systems are good for analytical work, she says, FPLC systems are
preferred for protein purification.

Anke Boerdgen, head of product management at Knauer, says another reason to choose FPLC rather
than HPLC is the sample itself. To maintain a protein in its native state, for instance, you cannot apply
high pressure, and you should work in a cool environment, like 4 oC, she saysconditions that are easily
achieved using FPLC.

FPLC systems

FPLC systems come in a variety of flavors, depending on application, automation and scale. GE
Healthcares KTA lab-scale systems are benchtop instruments for lab preparative work, for instance,
with different models that provide varying degrees of automation. Liljedahl says that for some FPLC
users, scalability is key. Once a protein of interest is established at the research scale, and a scale-up
process is developed, it is critical to take this to manufacturing, she says. KTA systems can support this
with models, such as KTApilot, a benchtop instrument for process development and manufacturing.
Larger models, such as KTAready and KTAprocess, support larger-scale production.
Bio-Rad Laboratories offers its new NGC Chromatography System in different models to suit various
applications, as well. Before building the system, says Richard Lee, lab chromatography marketing
manager at Bio-Rad, the company spent three years trying to understand customers needs, pain
points and the challenges that they are facing. The modular NGC is based on a core system, which
users then build on as needed.

Users choose from among six core systems, which differ in their degree of automation. The least
automated is the NGC Quest (or Quest Plus) system; it includes all the starting components for
purification, such as a pump, mixers, injector and detector. The NGC Scout (or Scout Plus) system
includes the Quest components as well as additional automation for method optimization. The NGC
Discover and DiscoverPro systems include the Scout system plus further automation, such as column
switching, automated sample loading and buffer selection. Lee says the NGC is also capable of other
automation tasks, such as tandem purification by linking multiple purification steps together into a single
continuous prep.

Researchers can load up to 15 columns per NGC system, and using an autosampler, screen up to 87
samples at a time, says Lee. The companys user-friendly NGC interface simplifies onboarding, especially
for novices. For example, the Point-to-Plumb feature integrates an LED light system and [a] flow path
thats displayed on the user interface, says Lee. It guides the user through tubing connections by
showing a flow diagram on-screen while also illuminating small, green indicator lights at the
corresponding connection sites. It helps customers to avoid costly plumbing mistakes," says Lee.
Also supporting FPLC is Knauer. The companys entry-level Azura Compact Bio LC 10, with flow rates of
up to 10 ml/min, is designed for basic applications, such as size-exclusion chromatography. Knauers Lab
Bio LC adds the ability to perform low- or high-pressure gradients and flow rates up to 50 ml/min,
depending on the modules chosen; the Pilot Bio LC can run at up to 800 ml/min, depending on the pump
heads selected.

Regardless of which Knauer system users choose, says Boerdgen, they have a choice of detection
systems, including variable single-wavelength UV, variable single-wavelength UV/VIS, variable multiplewavelength UV/VIS, diode array and fluorescence detectors.

FPLC applications
FPLC systems support a range of column types and sizes. GEs KTA systems, for instance, are
commonly used with affinity, ion-exchange, hydrophobic-interaction, multimodal and size-exclusion
chromatography, depending on the protein to be purified. Most used by academic customers are the
HiTrap columns in 1-ml and 5-ml [volumes], says Liljedahl.

Oftentimes, researchers combine separation strategies to effect more sophisticated purification schemes.
GE offers columns for affinity purification of proteins expressed with affinity tags, such as histidine and
GST tags. But, as a final polishing step in a purification protocol, we recommend that customers use our
size-exclusion chromatography columnssuch as the companys HiLoad Superdex, HiPrep Sephacryl or
Superdex/Superose columnsto get a size-homogeneous sample, Liljedahl says. To make selection
easier, GE offers a smartphone app called Purify to identify the best column types for specific
applications.
Bio-Rad's customers also typically combine protein purification and polishing, says Lee, and the
company offers affinity chromatography for polyhistidine- and protein A-tagged recombinant proteins as
well as ion-exchange or hydrophobic-interaction chromatography. Using automation and adding multiple
purification modules onto one NGC system, throughput can get a big boost, especially in drug discovery.
For instance, researchers can set up an affinity column followed by ion-exchange chromatography
followed by size-exclusion chromatography. Screen multiple samples through all three steps and then
come back the next day to review [the] data, Lee says. We can really configure the NGC in a multitude
of different ways, depending on what youre screening for.

Affinity, size-exclusion and ion-exchange chromatography are also very common among Knauer
customers, says Oliver Gueltzow, the companys head of online and print marketing. Due to a range of
switching components, we also can set up automated procedures, says Gueltzow. Common protein
types that Knauer customers purify include antibodies, enzymes and components of blood or cellular
lysates, according to Boerdgen. For applications that rely on purified proteins retaining their native
characteristics, Knauer also supplies cooling options. Knauer systems can be operated in a cold room,
says Boerdgen. And we offer a cooling system for our fraction collector and column so the system can
be operated in the general lab room, which is very convenient for the user.

With so many choices among FPLC features, protein purification is becoming faster, more productive
andabove allmore user-friendly. We came a long way in our ability to make things easy for the
researcher to purify the protein of interest, says Lee. At the end of the day, purification is a method to
get to the [product], and if you make the process as easy as possible, thats the desire of every
researcher out there.