Fast Protein Liquid Chromatography

Fast protein LC (FPLC) is a specialized form of HPLC that is designed for biomolecule purification and separation. The essential difference between HPLC and FPLC lies in the operating conditions. Protein structure cannot survive harsh conditions such as high pressure, high temperature, extreme pH or solvents typically used for HPLC.

As FPLC is designed to separate proteins, it utilizes lower pressure compared to regular HPLC and facilitates low-temperature operation. In place of solvents, FPLC uses buffers as its mobile phase in order to provide the optimum environment for protein separation.

Compared with HPLC and UHPLC, where pressures can usually go as high as 150 MPa, FPLC operates at lower pressures of 3–5 MPa. The lower pressure prevents the disintegration of protein structure while the sample is flowing through the column. Along with the low pressure, FPLC employs a higher flow rate of up to 100 mL/min to facilitate a larger sample volume.

In addition, proteins prefer low temperature to maintain their structure; the working temperature for proteins is usually around 4°C. Hence, FPLC instruments are often placed in a cold room with high humidity, and the components for FPLC need to have the durability to withstand this working environment.

Furthermore, the usage of buffers with high salt concentration to maintain protein structure would cause corrosion to stainless steel chromatography systems, and the metal ions from the stainless steel can interact with the protein and disturb its structure. In order to prevent these issues, FPLC is usually built with biocompatible materials, such as polyether ether ketone (PEEK) ceramic, titanium or carbon coating.

In terms of columns, FPLC has different size and packing materials compared to regular HPLC. HPLC columns are typically packed with silica beads, which have small particle sizes for high-pressure applications, while FPLC columns employ polymers/resins with larger particle sizes. Resins for FPLC are not as pressure resistant as silica beads and tend to be more sensitive to air bubbles. As pressure resistance is not an issue for FPLC, FPLC columns are usually made from transparent and biocompatible glass. With transparent columns, users can visually check the columns for air bubbles and monitor the stationary-phase condition during separations. Typical column chemistries utilized in an FPLC system include size exclusion, ion exchange, affinity and hydrophobic interaction chromatography.

FPLC was initially used only for protein separations; however, the wide variety of buffer and resins available for this technique has broadened its applications. In recent years, FPLC has found uses in life science academic research, especially proteome research. Biotechnology and pharmaceutical companies also utilize FPLC to purify monoclonal antibodies for antibody drug conjugates and other biotherapeutic applications.

The global FPLC market was estimated to be over $100 million in 2017,with a mid-single digit growth rate anticipated in 2018. The US and Europe led regional demand due to advanced life science research and a strong biotech and pharma sector in these regions. The Chinese government’s heavy investments in building its biotech industry has also created a high potential for FPLC demand in the country.

Currently, only a handful of vendors offer FPLC systems. GE Healthcare has upgraded its AKTA FPLC into AKTA Pure, equipped with a flexible and automated protein purification system. Bio-Rad Laboratories offers several models of its NGC and BioLogic systems. Knauer also competes in the FPLC market with its extensive Azura product line. ChromaCon, a smaller vendor, offers an FPLC system with its Contichrom product line. Large manufacturers such as Agilent Technologies, Shimadzu and Waters provide at least one bio-inert option in their HPLC product lines.

Leading Vendors:

  • GE Healthcare
  • Bio-Rad Laboratories
  • Knauer

Largest Market:

  • Academia
  • Biotechnology
  • Pharmaceuticals

Instrument Cost:

  • $20,000–$50,000
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