Integrated acelluar pertussis vaccine purification process development by using manufacturing process optimization

Abstract

Pertussis, also known as whooping cough, is a highly contagious respiratory disease transmitted directly between humans. Pertussis is caused by the bacterium Bordetella pertussis and can affect people of all ages, with serious or even deadly outcomes for babies under one year of age. The whole-cell pertussis (wP) vaccine developed in the late 1940s protected against pertussis and rapidly reduced incidence. However, adverse reactions such as febrile convulsions and , very rarely, hypotonic-hyporesponsive episodes were observed at a high frequency after administration of the wP vaccine, underlining the need for a safer pertussis vaccine. Acellular pertussis (aP) vaccine composing thress components including such as pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) from B. pertussis, is safer and less reactogenic than the wP vaccine. However, due to difficulties in the manufacturing process of acellular pertussis vaccines, only two acellular pertussis vaccines are approved and sold. The purpose of this study is to develop a manufacturing process that overcomes the major technical limitations of the PT, FHA, and PRN antigen manufacturing process. Three types of antigens can be obtained by Bodetella pertussis cell culture, PRN can be obtained from cell pellets and PT and FHA can be obtained from culture supernatant. PRN, a non-fibrial outer membrane protein of Bordetella pertussis, is a limiting component of cell-free pertussis vaccine due to its low expression, so the study of PRN manufacturing process was conducted with the main goal of improving productivity. We compared the extraction of PRN from the cell membrane using urea with the commonly used heating method and found that more membrane proteins were extracted using the urea method. Through the study of various conditions, it was found that the cell pellet processing conditions, including freezing and thawing, substantially affect the PRN yield. Finally, a single cycle of rapid freezing of the cell pellet with slow thawing resulted in up to 5-fold higher PRN yields than conditions without freezing and thawing. We also explored urea treatment conditions and found the optimal conditions. After extraction, residual impurities were removed by sequential anion exchange, hydrophobic interaction, and gel filtration chromatography to obtain highly purified PRN antigen with a purity of more than 95%, thus establishing a PRN manufacturing process. PT and FHA are expressed in relatively large amounts and are obtained from the culture supernatant. Due to its strong toxicity, PT must undergo a detoxification process after the purification process. Because only PT undergoes detoxification process, cross-contamination of PT and FHA may cause safety concerns during vaccine manufacturing. So it is necessary to completely separate PT and FHA, and conditions capable of completely separating the two antigens were established through optimization of affinity column chromatography conditions. The design of experiment(DoE) method was used to optimize the conditions, and a design space capable of stably isolating antigens was selected. Through sequential hydroxyapatite, hydrophobic interaction, and affinity chromatography processes, PT and FHA antigens with a purity of more than 95% were obtained. Scale-up study was conducted up to 200 L scale and reproducible results are confirmed. Acellular pertussis vaccine for adults was prepared by mixing three types of PT, FHA, and PRN antigens produced from a 200 L scale with diphtheria toxoid and tetanus toxoid, and its safety and efficacy were confirmed through non-clinical and clinical studies. In conclusion, an integrated acelluar pertussis vaccine purification process for three types of antigens from Bodetella pertussis culture was developed. The membrane protein extraction process using urea developed in this study may also serve as a reference for manufacture of other membrane proteins and the methodology for finding antigen separation using affinity chromatography can be applied to the production of a variety of proteins. In addition, this pertussis antigen manufacturing method will be applicable to the development of various combination vaccines based on acellular pertussis.