Efficient Purification Of The Pertussis Antigens Toxin, Filamentous Haemagglutinin, And Pertactin In Chromatography Workflows

Bordetella pertussis is a Gram-negative bacterium causing whooping cough. There are two types of pertussis vaccines available: whole-cell vaccines and acellular vaccines (purified components). While whole-cell pertussis vaccines are commonly associated with adverse reactions, acellular pertussis vaccines exhibit fewer side effects and, hence, constitute a viable alternative to whole-cell pertussis vaccines for routine use in disease prevention. Acellular vaccines can be based on different immunogens such as detoxified PT, FHA, or PRN. This work aims to demonstrate a purification process for PT, FHA, and PRN with a purity of over 95% for each antigen. The antigens purified using the described method can be used in either acellular pertussis stand-alone vaccines or in combination vaccines.

A method comprising two-fold salt precipitations followed by a sucrose gradient centrifugation step is sometimes used in purification of PT, FHA, and PRN. This purification strategy suffers from poor separation of impurities from the target molecules, resulting in low purity and yield. In addition, the process cannot separate the different targets, resulting in a mixed product without possibility to control antigen ratios. The process is also difficult to scale to meet market demands.

The purification strategy presented here is based on modern, scalable chromatography resins (Fig 1). For both PT and FHA, Capto™ SP ImpRes cation exchange resin was used in an initial capture step. Capto SP ImpRes is a strong cation exchange resin. The resin offers high resolution and excellent pressure-flow properties for fast and efficient purification of biomolecules. For PT, a second polishing step was added in which Capto MMC resin was used. Capto MMC is a multimodal cation exchange resin that can be used to bind proteins at the conductivity of the feed material and is commonly used to solve more difficult purification challenges.