Development of one-step purification process to analyze the quality of the culture media
- 주제(키워드) purification , process , onestep
- 발행기관 아주대학교
- 지도교수 서민덕
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030170
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This "One-step" study is trying to develop a protein coupled affinity purification which is instead of a number of conventional processes for the quality analysis of items in the recombinant glycoprotein culture fluid currently in production. Here, We purification processes by coupled with based resin through recombinant glycoprotein antibodies applying antibody-antigen- interactions and observing for many glycan to recombinant glycoprotein of using glycan-targeting resins (Immobilized D-galactose gel, HA ultragel, Con-A sepharose FF) used it. We estimated the process that has good performance by comparing with the existing process through the process. The coupling process to based resin with protein was confirmed. In contrast, to its confirmed coupling resin use purification process did showed a low affinity in the purification process of recombinant glycoprotein that was other affinity tested. The other glycan-targeting resins are Immobilized D-galactose gel without to detected D-galactose in protein structures and the HA ultragel or Con-A sepharose FF 4B resins were low capacity ligand interactions with proteins. Thus, The purification process for recombinant glycoprotein was compared with the traditional purification process using three resins that are glycan-targeting and coupled with recombinant glycoprotein antibody resin.
more초록/요약
본 "One-step" 연구는 현재 생산 중인 recombinant glycoprotein 배양액에서 품질분석을 위한 기존 다수의 정제 공정을 대신하는 단백질과 coupled affinity 정제를 이용하여 개발을 진행하였다. 여기서는 antibody-antigen 상호작용을 응용하여 recombinant glycoprotein과 based resin과 coupled 하여 정제 공정을 진행하였고 recombinant glycoprotein에 glycan이 다수 관찰되는 것을 이용한 glycan-targeting 레진들 (Immobilized D-galactose gel, HA ultragel, Con-A sepharose FF)를 사용하였다. 이 공정을 통해 기존 공정보다 좋은 performance를 기대하였다. 단백질과 resin과의 결합은 확인되었다. 이와는 대조적으로, 기존의 알려진 정제 공정과 다르게 affinity test에서 recombinant glycoprotein 이 낮은 affinity를 확인하였다. 다른 glycan-targeting resin은 단백질 구조에서 D-galactose가 immobilized D-galactose gel에 검출되지 않았고, HA ultragel과 Con-A sepharose FF 4B resin들은 단백질과 ligand 상호작용의 capacity가 낮았다. 따라서 커플링 공정으로 항체화한 단백질과 결합한 resin과 glycan-targeting하는 4가지 resin을 이용하여 recombinant glycoprotein에 대한 정제 공정을 기존의 정제 공정과 비교하였다.
more목차
Ⅰ. Introduction 1
Ⅱ. Materials and methods 4
2.1 Materials 4
2.2 Apparatus 4
2.3 Methods 5
2.3.1 Downstream process for recombinant glycoprotein antibody coupled with base resin 5
2.3.1.1 Purification of recombinant glycoprotein antibody serum 5
2.3.2 Coupling to base resin with recombinant glycoprotein antibody 6
2.3.2.1 Purification of cell culture with coupling affinity resin 7
2.3.3 Refining the culture fluid using resin holding the glycan 7
2.3.4 Compare with chromatography process 8
Ⅲ. Result and discussion 9
3.1 Downstream process for recombinant glycoprotein with base resin 9
3.1.1 Purification of recombinant glycoprotein antibody serum 9
3.1.2 Coupling with recombinant glycoprotein antibody and base resin 12
3.1.3 Coupling resin purification chromatography 14
3.1.3.1 Concentration of cell culture broth 14
3.1.3.2 1st purification of recombinant protein cell culture 14
3.1.3.3 2nd purification of recombinant protein DS 16
3.1.3.4 3rd purification of recombinant protein DS 18
3.1.3.5 4th purification of recombinant glycoprotein DS 20
3.1.3.6 Coupling identification and analysis about coupling resin 22
3.2 Purification using glycan-targeting resin 24
3.2.1 1st purification using Immobilized D-galactose gel 24
3.2.2 HA ultragel using purification 26
3.2.2.1 1st purification using bind-elute mode of HA ultragel 26
3.2.2.2 1st purification using flow-through mode of HA ultragel 28
3.2.3 1st purification using 1st Con-A sepharose 4B FF resin 30
3.2.4 Purification using resin glycan-targeting with recombinant glycoprotein DS 32
3.2.4.1 Buffer replacement about recombinant glycoprotein DS 32
3.2.4.2 2nd purification using Immobilized D-galactose gel 32
3.2.4.3 2nd purification using 2nd Con-A sepharose 4B FF resin 34
3.2.4.4 2nd purification using 2nd HA ultragel bind-elute mode 36
3.3 Discussion 36
Ⅳ. Conclusion 38
Ⅴ. Reference 39
