Process Optimization for Production of Tetanus and Diphtheria Toxoid Vaccines
- 주제(키워드) diphtheria toxin , tetanus toxin , toxoid , fermenter , Td vaccine
- 발행기관 고려대학교 대학원
- 지도교수 김익환
- 지도교수 최용경
- 발행년도 2016
- 학위수여년월 2016. 8
- 학위구분 박사
- 학과 대학원 생명공학과
- 원문페이지 105 p
- 실제URI http://www.dcollection.net/handler/korea/000000068556
- 본문언어 영어
- 제출원본 000045881532
초록/요약
Diphtheria and tetanus toxoids are an antigen in the Td vaccine. Diphtheria toxin was produced by culturing Corynebacterium diphtheriae in a fermenter in high expression, followed by purification and detoxification. Specific cultivation profiles of pH and dissolved oxygen were identified that resulted in the production of high level expression of diphtheria toxin in the fermenter. Production of 300 Lf/ml or higher of diphtheria toxin in a fermenter was only possible when using a specific pH profile, and the production process was divided into four stages. Diphtheria toxoid after detoxification by formaldehyde was purified to a purity level of 2500 Lf/mgPN or higher. The purified diphtheria toxoid was used as the antigen of Td vaccine in clinical trials. Clostridium tetani was cultivated in a fermenter and a single-use bioreactor. In order to produce tetanus toxin by cultivating C. tetani, N-Z-Case TT has to be autoclaved with glucose, and autolysis of C. tetani should occur so that the toxin can be secreted into the medium. Tetanus toxoids produced from the fermenter and the single-use bioreactor were equivalent and showed purities of 2500 Lf/mgPN or higher. The tetanus toxoids produced in the fermenter and single-use bioreactor were used as the antigen of Td vaccine in clinical trials.
more목차
Chapter I. Production of highly pure toxoid from diphtheria
toxin through optimized fermenter cultivation of
Corynebacterium diphtheriae…..……….…..…….. 1
1. Abstract ……………………………………………….… 2
2. Introduction …………………………………………….. 3
3. Materials and Methods ………………………………… 6
3.1 Bacterial strain …………………………………..…..…..…… 6
3.2 Preparation of N-Z-Case Plus solution ……………...……..… 6
3.3 Preparation of modified Mueller’s growth factor solution ....... 6
3.4 Media for cultivation …………………………………............ 7
3.5 Seed culture preparation ………………………………........... 7
3.6 Production of toxin and toxoid …………………...…..……… 8
3.7 Maltose measurement ………………………………….......… 9
3.8 Measurement of limes flocculation (Lf) …………….............. 9
3.9 Nitrogen determination …………...………….…………..…. 10
3.10 KLa measurement ..………………………….…..………..… 10
3.11 SDS-PAGE ………………………………….………………10
3.12 Immunodiffusion …………………………….…………….. 11
4. Results ………………………………………………….. 12
4.1 Conditions for the optimal diphtheria toxin expression …...... 12
4.2 Variations of the fermentation parameters .……………..…....14
4.3 Shift in toxin expression conditions …...………………..…... 21
4.4 Production of diphtheria toxoid ……………………….…….. 23
5. Discussion ……………………………….…….……….. 28
6. References …...…………………………………………. 42
Chapter II. Comparison of Clostridium tetani cultivated in
the fermenter and the single-use bioreactor from
toxin production to toxoid purification ......…… 47
1. Abstract ………………………………………………… 48
2. Introduction ……………………………………………. 49
3. Materials and Methods ………………………………... 52
3.1 Bacterial strain …………………………………….………… 52
3.2 Preparation of N-Z-Case TT solution …………………….…. 52
3.3 Preparation of stock solution for medium ..………….……… 52
3.4 Media for toxin production ………………………………….. 53
3.5 Seed culture preparation.…………………………………….. 54
3.6 Production of toxin and toxoid ……………………………… 54
3.7 Culture analysis …………………………………………...… 56
4. Results ……………………………………………….…. 58
4.1 Production of tetanus toxin in the fermenter and the single-use
bioreactor ……………………………………………………. 58
4.2 Tetanus toxin production conditions ………………………… 63
4.3 Level of purification of tetanus toxoid ……………………… 66
4.4 Comparison of the physical and chemical properties of the
tetanus toxoids ….…………………………………………… 68
5. Discussion ………………………………………………. 75
6. References ……………………………………………… 85
Concluding Remarks …...……….…..……………………... 89
Abstract in Korean …………………….…………………... 91
