Production of Erythropoietin by L-132, a Lactate Consuming Human Cell Line
- 발행기관 고려대학교 대학원
- 발행년도 2004
- 학위명 박사
- 학과 대학원:생명공학과
- 식별자(기타) DL:000014914273
- 서지제어번호 000045213498
초록/요약
Lactate and ammonia are two toxic waste products formed during mammalian cell cultures. Accumulation of these side products has negative effects on the cell growth and protein production. Among various animal cell lines, L-132 (ATCC CCL-5) cells were known to be adapted to glucose-free medium. The adapted cells were able to consume pyruvate as a carbon source of energy with the accumulation of low level of lactate. In addition, lactate accumulated in the early period of cell culture were consumed in the late period. In this study, L-132 cells were used as the host cell of recombinant human erythropoietin. Epo gene was amplified and expression of EPO was increased by gene amplification in increased MTX concentration. To improve EPO productivity, single cloning selection was carried out through limiting dilution. Clonal variation in growth rate and production was observed. Our data suggest that the characters of lactate consuming and pH controlling has advantage on the cell growth and EPO production. EPO production level was reached about 1000U/mL. Taken all together, L-132 cells can be used as a useful human cell line for the high level production of recombinant proteins.
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Abstract........................................................................i
Table of Contents........................................................iii
List of Figures...............................................................v
List of Tables...............................................................vii
List of Abbreviations......................................................viii
Introduction...................................................................1
Materials and Methods...................................................6
1. Transfection and Selection of the Cells......................6
2. Cells and Medium...................................................6
3. Amplification of epo gene.........................................8
4. Single Cell Cloning..................................................8
5. Identification of EPO mRNA level by RT-PCR.............10
6. Counting Viable Cells..............................................10
7. Analyses of Medium Metabolites..............................11
8. Bioassay for Quantitative Analysis of EPO.................11
Results and Discussion.................................................13
1. CHO and L-132 Cells..............................................13
1.1 Comparison of CHO and L-132 cells...................13
2. Amplification of EPO Gene of L-132 Cells..................18
2.1 MTX concentration for adaptation of L-132 cells....18
2.2 r-L132 adaptation in medium containing MTX........21
2.3 Identification of EPO gene amplification................26
3. Selection of single clones for higher EPO production...31
3.1 Single clonal selection by limiting dilution.............31
3.2 Characterization of single clones.........................33
4. Amplification of EPO gene of r-L132 cell lines in the
absence of selective pressure..................................39
4.1 Comparison of growth and EPO production o
