Advancing Protein Purification: The Evolution and Industrial Impact of FPLC Chromatography

Chromatography is a vital tool in protein purification, offering precise and efficient separation based on various protein properties.

Chromatography is one of the most effective techniques to achieve this goal, offering high resolution, scalability, and versatility. Protein Purification isolates specific proteins from cells, tissues, or biological fluids, removing impurities like DNA, host cell proteins, and aggregates. It is essential for producing high-purity therapeutic proteins, vaccines, and diagnostic tools, as well as studying protein structure and function. 

There are several types of chromatographic techniques commonly used in protein purification. One in those is Fast Protein Liquid Chromatography (FPLC).

Fast Protein Liquid Chromatography (FPLC)

Often FPLC is widely used for protein purification and characterization. It offers high resolution and reproducibility, preserving their native structures. This technique features high loading capacity, biocompatible buffer systems, fast flow rates, and stationary phases for common chromatography modes (e.g., gel filtration, ion exchange, reversed phase, and affinity chromatography).

FPLC accommodates the users to monitor several parameters at a time such as UV level, pH, and conductance and it allows for multiple columns to be run in tandem. Therefore, in overview of minimizing the time needed to isolate pure protein. The method is applicable to proteins as well as other kinds of biological samples including oligonucleotides and plasmids.

FPLC -separates, purifies, and analyses protein mixtures based on their physical and chemical properties (size, charge, hydrophobicity, or ligand binding). Fast Protein Liquid Chromatography (FPLC) is a medium-pressure liquid chromatography technique designed to purify and analyse proteins and other biomolecules .

It operates at low pressures using liquid mobile phases (buffers) and solid stationary phases (resins) to separate biomolecules efficiently, often in cold rooms to maintain protein stability. 

FPLC relies on different chromatographic methods:

Ion Exchange (IEX): Separates proteins based on their net surface charge.

Size Exclusion (Gel Filtration): Separates proteins based on their molecular size.

Affinity Chromatography: Separates based on specific, reversible binding affinity to a ligand.

Liquid Chromatography Setup: A pump pushes a buffer (mobile phase) through a column packed with stationary phase resin.

Pressure & Speed: FPLC operates at lower pressures than HPLC, allowing for the use of soft, gel-based resins ideal for delicate proteins.

Monitoring & Detection: The system uses detectors for UV (typically 280 nm for proteins), conductivity, and pH to monitor separation in real-time.

Automated Fraction Collection: The system automatically collects separated proteins into different tubes for further analysis. 

Core Principle

The fundamental principle of FPLC is the differential partitioning of sample components between a stationary phase (typically a resin or gel beads) and a mobile phase (a liquid buffer). As the mobile phase is pumped through the column at a controlled rate, different proteins interact uniquely with the stationary phase based on their physical and chemical properties. 

In Fast Protein Liquid Chromatography, the solvent velocity is controlled by a microprocessor through a software interface so as to maintain the constant flow rate of the solvents. The eluant is passed through the detectors to measure the salt concentration (by conductivity) and protein concentration (by absorbing ultraviolet light at a wavelength of 280nm).

The speed at which a protein travels through the column and its retention time is thus determined by the strength of the interactions. Proteins with weaker interactions elute (exit) the column first, while those with stronger interactions elute later. 

Apparatus & Equipment

The FPLC system consists of a control unit, high-precision pumps, a column, detection system, and a fraction collector. The pump allows the fraction of each buffer entering the column to be continuously varied. The injection loop, a segment of tubing of known volume, is filled with the sample solution to be injected in the column. The sample loading is done by the injection valve which links the mixer and sample loop to the column. The FPLC column is a glass or plastic cylinder packed with beads of resin. It is mounted vertically with the buffer flowing downward from top to bottom. The eluant from the column passes through the flow cells for protein concentration measurements. The detector records the salt and protein concentration.

FPLC modes

Ion-exchange FPLC

1. Prime the pumps A and B with filtered and degassed buffers A (10 mM Tris–HCl, pH 7.0.) and B (10 mM Tris–HCl, pH 7.0, 1 M NaCl), respectively.
2. Set the pressure limits on both the pumps below the maximum for the column in use.
3. Equilibrate the Mono Q column (1 mL volume) with 5 volumes of buffer A and 10 volumes of buffer B and then with 5 volumes of buffer A.
4. Wash the sample loading loop with buffer A.
5. Load the sample (0.5–10 mL) (approximately 1 mg/mL) and wash the column with buffer A. Collect the flow-through and evaluate for the protein of interest.

Note: If protein is not bound then replace the Mono Q column with the Mono S column.

• Regenerate the column by washing it with 10 volumes of buffer B, then with 5 volumes of buffer A.

Scouting FPLC methods

1. Create gradients of shallowness by varying the concentration of buffer B at different time-points.
2. Identify the column to which the protein binds and perform chromatography at different pH values using various buffer systems.

Strengths and limitations

• The fast-protein liquid chromatography is a simple and reproducible separation technique with efficient resolution.
• The chromatography columns have longer lifetime because of the inert construction against the high salt concentrations and corrosive liquids.
• The FPLC supports a wide range of columns as the procedures are carried out under low pressure.
• The wide flow range makes it a suitable technique for analytical and preparative Chromatography.
• The technique needs glass columns and cannot withstand high pressures.
• The method is not sensitive to thermolabile proteins.