Suspension CHO cell용 대량 단백질 발현 시스템CHOgro® Expression System
(Code MIR 6260)
•셋업이 간편: 대량 단백질 생산에 최적인 프로토콜 제공
•Cell clumping 없이 균일한 단백질 발현 및 높은 viability 실현
•Animal origin free, hydrolysate-free•
•cGMP (국제의약품 품질 기준) 조건에서 생산대량 단백질 생산에 최적Transfection 시약과 배지가 모두 포함된 All-in-One Kit
Figure 1. Suspension CHO cells grow to high density in the CHOgro® Expression Medium. Trip-licate flasks of FreeStyle™ CHO-S cells were seeded in CHOgro® Expression Medium (red line)or FreeStyle™ CHO Expression Medium (blue line) at a cell density of 0.5 x 106 cells/㎖. Cellcounts (solid line) and viability (dotted line) were measured daily using a Guava easyCyte™5HT flow cytometer.
Figure 2. CHOgro® Expression Medium yields multi-fold increases in antibody titer. HumanIgG1 was produced by transient transfection using TransIT-PRO® (1:1), FreeStyle™ MAX(1.25:1.25) or 25kDa linear PEI (6:1) transfection reagents according to the manufacturers’or published protocol (reagent:DNA ratio).
FreeStyle™ CHO-S cells were transfected using 1㎍plasmid DNA per milliliter of culture and cell densities of 2 x 106 cells/㎖ or 1 x 106 cells/㎖ forthe CHOgro® Expression Medium (red bars) or FreeStyle™ Expression Medium (blue bars).
Antibody levels were also analyzed from day 6 clarified supernatants using a human IgG ELISA.Expression system에 따른 단백질 발현 비교간편한 culture system 변경과 발현율 증가CHOgro® Cell DensityViability (%, dotted lines)Milion cell / mililiter (solid lines)Day post seedingCHOgro® ViabilityFreeStyle™ CHO Cell DensityFreeStyle™ CHO ViabilityAntibody Titer (%)Normalized to CHOgro®systemTransfection Reagent:CHO-S Growth Media:Freestyle™ MaxFreestyle™ CHOTransIT-PRO®Freestyle™ CHOTransIT-PRO®CHOgro®TransIT-PRO®24hr adaptationto CHOgro®단백질 발현 상승IgG (㎎/L)CHOgro® Expression MediumTransIT-PRO® (1:1)FreeStyle™ Max (1.25:1.25)25kDa Linear PEI (6:1)FreeStyle™ CHO Expression Medium A.
Day 0 Day 1
Highertiters Dilute cells andtransfectExchange mediato CHOgro®Dilute cells andtrasfectCHO-S cells grown inFreestyle™ CHO mediaCHO-S cells grown in CHOgro® media구성품TransIT-PRO® Transfection ReagentCHOgro® Expression MediumCHOgro® Complex Formation SolutionPoloxamer 188L-Glutamine용량1 ㎖1ℓx 2100 ㎖100 ㎖100 ㎖Figure 3. Media exchange leads to higher protein production.FreeStyle™ CHO-S cells were cultured in FreeStyle™ CHO Expres-sion Medium or CHOgro® Expression Medium and 24 hours prior totransfection a subset of the cells grown in FreeStyle™ CHO Expres-sion Medium were spun down and exchanged with 100% fresh CHO-gro® Expression Medium. The cells were allowed to grow and adaptfor 24 hours prior to transfection with FreeStyle™ MAX or Tran-sIT-PRO® transfection reagents according to the manufacturers’protocol and a hIgG1 encoding construct. (A) Workflow schematicof media exchange of CHO-S cells from FreeStyle™ CHO ExpressionMedium to CHOgro® Expression Medium (black arrow) or the nor-mal CHOgro® Expression System (red arrow) (B) Day 6 supernatantswere clarified and analyzed using a human IgG ELISA. Data is nor-malized to the complete CHOgro® Expression System (red bar).Media에 따른 cell density, viability 비교ver. 201601
Transfection & Endotoxin Removal KitTransIT-PRO® Transfection Kit (Code MIR 5700)
•Suspension CHO와 293 cell 용 transfection reagent•다양한 CHO 배지 조성에서 호환성 우수
•다양한 culture volume (4-400 ㎖)에 적용 가능Transfection 후 시간경과에 따른 발현율 비교고객이 직접 경험한 TransIT-PRO® 높은 발현내 Cell, Mouse가 아파~E.coli 증식시 오염된 Endotoxin 때문에…Transfection이 잘 되었나?
• Label IT® Plasmid Delivery Control, Cy® 3 (Code MIR 7904)
• Label IT® Plasmid Delivery Control, Fluorescein (Code MIR 7906)Day 3Day 5Day 7Human Fc (μg/L)TransIT-PRO® +PRO Boost Reagent400300200100025kDa Linear PEIFreeStyle™ MaxTime post transfection (Day)Fluorescence per 1E + -05 viable cells(Arbitrary unit/E + 05 cells)Lipofectamine 2000 (37 ℃)TransIT-PRO (37 ℃)PEI (37 ℃)Lipofectamine 2000 (32 ℃)TransIT-PRO (32 ℃)PEI (32 ℃)TransIT-PRO® Transfection Kit 구성구성품TransIT-PRO® Transfection ReagentPRO Boost Reagent용량1 ㎖1.5 ㎖Figure 4. Achieve high antibody titers using the TransIT-PRO® Transfection Kit. Human IgG1was produced by transient transfection using TransIT-PRO and PRO Boost Reagent, 25kDalinear PEI or FreeStyle™ Max transfection reagents according to the manufacturers or pub-lished protocol. Transfections were performed using 1㎍ plasmid DNA per milliliter of cultureand 0.5 x 106 cells/㎖ at the time of transfection. FreeStyle™ CHO-S cells were cultured in 20㎖ of FreeStyle™ CHO Expression medium in 125 ㎖ shake flasks. Day 3, 5 and 7 supernatantswere clarified and analyzed using a human IgG-Fc sandwich ELISA.Figure 5. Evaluation of EGFP-C3 protein expression level upon transient transfection withdifferent reagents. EGFP-C3 protein expression per viable cell at 37 °C & 32 °C.[인용 논문. Evaluation of transfection methods for transient gene expression in Chinesehamster ovary cells. Sou S, et al. ABB, 2013 Dec. Vol. 4.]MiraCLEAN® Endotoxin Removal Kit(Code MIR 5910)• Plasmid의 E.coli endotoxin 제거로단백질 발현 향상• 두 종의 reagent 첨가로 간편하게 제거• in-vitro, in-vivo 모두 적용 가능(in-vivo 시 면역반응 감소)Endotoxin 제거를 통한 단백질 발현 향상Luciferase Expression (pg)Commercial Plasmid(35 EU/mg)Crude plasmid(11,635 EU/mg)Crude Plasmid with 3 Rounds ofMiraCLEAN Treatment(10 EU/mg)Figure 6. Plasmid DNA exhibits high transfection efficiency after treatmentwith the MiraCLEAN® Kit. COS-7 cells were transfected with 2 ㎍ of plasmidDNA and 2 ㎕ of the TransIT®-LT1 Reagent in 6-well plates. Cells were har-vested at 24 hours post transfection and assayed for luciferase expression.
EU=Endotoxin Units발현율증가고 객 (제품문의)02-2081-2510지원센터