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Purification of proteins in cell culture processes

Protein purification refers to the series of processes that help separate one or multiple proteins from a complex mixture. These purification processes enable the characterization of protein function and structure or the production of target proteins for biopharmaceutical applications. Protein purification leverages attributes such as size, shape, binding capacity, total ionic charge, and hydrophobic groups. The two broad categories of protein purification are:

  • Analytical purification: These purification techniques produce moderate amounts of protein isolates for research or analytical purposes.
  • Preparative purification: Preparative techniques produce large quantities of purified proteins, such as monoclonal antibodies.

Chromatographic separation

Chromatography is one of the main ways proteins and other biomolecules are purified from mixtures. In liquid chromatography, a liquid containing the target molecule, such as a cell lysate, is passed over a stationary phase that helps separate the target molecule based on polarity, binding affinity, or other characteristics.

Types of chromatography

Chromatography systems come in many different sizes with different scales and purposes. The common methods of chromatography include ion-exchange chromatography, size exclusion chromatography, affinity chromatography, reverse phase chromatography, and hydrophobic interaction chromatography. For all of these methods, chromatography monitors provide data on factors that affect the reaction or indicate product quality, such as sample pH, conductivity, temperature, and optical density.

Relevance of pH in chromatography

The pH of the mobile phase is a crucial parameter that affects the process reaction and target biomolecule retention. The pH can change in varying ways during this phase, especially after injection of a sample. A mobile phase can also absorb CO2 on standing, which makes it more acidic. pH is, therefore, important in chromatography, since the retention time of sample components may change with varying pH. It is therefore important to track real time pH changes using chromatography monitors.

Chromatography monitors FAQs

Here are answers to some frequently asked questions about chromatography monitors.

What is a chromatography monitor?

Chromatography monitors or sensors are tools that provide real-time measurements of properties of a solution that are important for a chromatographic separation. Such properties include temperature, pH, conductivity, and optical density (OD) or absorbance at a given wavelength in the ultraviolet (UV) or visible spectrum.

What is a pH monitor?

A pH monitor or pH sensor is a chromatography monitor that measures the pH of a sample during a chromatographic run.

What is a UV monitor?

UV monitors or detectors measure the optical density of a mixture during a chromatographic separation. Optical density and/or absorbance can be used to determine the purity of a given sample.

What is a conductivity sensor?

Conductivity sensors are chromatography monitors that measure the conductivity of a sample or buffer in the flow path. These may be present in the flow path before or after the separation column.