목차

1. 서론 1
1.1 생물의약품(Biopharmaceuticals) 1
1.1.1 생물의약품 1
1.1.2 동등생물의약품(Biosimilars) 2
1.2 동물세포배양 6
1.2.1 동물세포배양 6
1.2.2 Chinese Hamster Ovary(CHO) cell 8
1.2.3 재조합 단백질 발현 체계 9
1.3 재조합 단백질(Recombinant protein) 10
1.3.1 유전자 재조합 기술 10
1.3.2 Erythropoietin(EPO) 14
1.3.3 Albumin-EPO(Alb-EPO) fusion protein 15
1.4 CHO 세포 배양에서 단백질 생산성 증진 방법 18
1.4.1 SGH 18
1.4.2 Anti-apoptotic agent 19
2. 연구 목적 21
3. 재료 및 방법 22
3.1 세포배양 및 배양조건 22
3.1.1 세포주 22
3.1.2 세포배양 22
3.1.3 첨가물질 22
3.2 분석 23
3.2.1 세포 수 및 생존도 측정 23
3.2.2 배지 내 Alb-EPO의 정량분석 23
3.2.3 Metabolite 분석 24
3.2.4 Cell cycle 분석 24
3.2.5 Cell death 분석 : Apoptosis/Necrosis 26
4. 결과 및 고찰 27
4.1 회분식 배양에 의한 Alb-EPO 생산 세포의 특성 27
4.2 SGH에 의한 Alb-EPO 생산 세포의 생산성 32
4.2.1 SGH 농도에 따른 세포의 생장 32
4.2.2 SGH 농도에 따른 Alb-EPO 생산성 32
4.2.3 SGH 농도에 따른 세포 주기의 분석 34
4.3 SGH와 anti-apoptotic agent과의 비교 분석 37
4.3.1 Anti-apoptotic agent에 따른 세포 생장의 분석 37
4.3.2 Anti-apoptotic agent에 따른 세포 주기의 분석 38
4.3.3 Anti-apoptotic agent에 따른 세포의 apoptosis/necrosis 분석 41
5. 결론 45
6. 참고문헌 47
그림목차
Figure 1. DHFR-mediated gene amplification in CHO cells. 12
Figure 2. Commercial EPO products. 16
Figure 3. Advantage of albumin fusion technology. (A) Increase of half-life in vivo and (B) useful of design flexibility. 17
Figure 4. Structure of SGH in Silkworm. 19
Figure 5. Time course change of (A) viable cell density and (B) viability in CHO cell batch cultures. 29
Figure 6. Time course change of (A) glutamine, ammonia concentration and (B) glucose, lactate concentration in CHO cell batch cultures. 30
Figure 7. Alb-EPO production in batch cultures. 31
Figure 8. Effect of SGH on (A) viable cell density and (B) viability. Cells were initially cultivated at without SGH (control, ) for 4 day and then cultivated at 0.5 mg/mL SGH addition (), 0.75 mg/mL (), 1 mg/mL (), 1.25 mg/mL (). 33
Figure 9. Effect of SGH on Alb-EPO production. Cells were initially cultivated at without SGH (control, ) for 4 day and then cultivated at 0.5 mg/mL SGH addition (), 0.75 mg/mL (), 1 mg/mL (), 1.25 mg/mL (). 35
Figure 10. Effect of SGH on cell cycle at day 6. 36
Figure 11. Effect of various anti-apoptotic agents on (A) viable cell density and (B) viability. Cells were initially cultivated without anti-apoptotic agents (control, ) for 4 day and then cultivated at 1 mg/mL SGH addition (), 2.5 mM dextran sulfate (), 8 mM NAC (). 39
Figure 12. Effect of various anti-apoptotic agents on Alb-EPO production. Cell were initially cultivated without anti-apoptotic agents (control, ) for 4 day and then cultivated at 1 mg/mL SGH addition (), 2.5 mM dextran sulfate (), 8 mM NAC (). 40
Figure 13. Effect of anti-apoptotic agents on cell cycle at day 6. 42
Figure 14. Effect of anti-apoptotic agents on cell death: apoptosis, necrosis at (A) day 5 and (B) day 6. 44
표 목차
Table 1. Top classes of biopharmaceuticals with 2010 sales 3
Table 2. Patent expiry of blockbuster biopharmaceuticals 5
Table 3. EU biosimilars approvals, status 2010 7
Table 4. FDA approved recombinant proteins 13
Table 5. Buffer composition for ELISA 25