Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200...

Area of Science:

• Biotechnology
• Immunology
• Biochemistry

Background:

• Tetanus purified anatoxin is crucial for immunoprophylactics.
• The World Health Organization (WHO) mandates high purity standards (≥1,000 Lf/mg protein nitrogen) for tetanus anatoxin.
• Liquid chromatography is a common but complex method for tetanus anatoxin purification at production scale.

Purpose of the Study:

• To develop and validate a scalable, high-yield purification method for tetanus anatoxin.
• To combine tangential flow filtration (TFF) with gel filtration for large-scale purification.
• To ensure the purified tetanus anatoxin meets WHO purity specifications.

Main Methods:

• Small-scale purification of tetanus anatoxin using Sephacryl S-200 High Resolution gel filtration.
• Large-scale purification combining tangential flow filtration (TFF) with a 50,000 N.M.W.L. ultrafiltration membrane and Sephacryl S-200 High Resolution gel filtration.
• Utilizing the Bioprocess System (Amersham Pharmacia Biotech) for purification.

Main Results:

• Successful high-yield purification of tetanus anatoxin was achieved on a small scale using Sephacryl S-200.
• The combined TFF and gel filtration method was scaled up effectively.
• 14 consecutive lots of tetanus anatoxin were purified, yielding 77,401,332 doses of tetanus toxoid.

Conclusions:

• The presented method, combining TFF and Sephacryl S-200 gel filtration, is reproducible and reliable for large-scale tetanus anatoxin purification.
• The method consistently meets WHO purity requirements for tetanus anatoxin.
• This scalable purification process supports the large-scale production of tetanus toxoid immunoprophylactics.