Consistent Production of Genetically Stable Mammalian Cell Lines - The author describes expression technology that produces cell lines with high genetic stability. This article is part of a special se

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Consistent Production of Genetically Stable Mammalian Cell Lines
The author describes expression technology that produces cell lines with high genetic stability. This article is part of a special section on expression systems.


BioPharm International
Volume 25, Issue 5, pp. 56-59

DEVELOPMENT AND CHARACTERIZATION OF GPEX LINES


Table I
The data presented in this article are from a number of GPEx generated cell lines producing various products. Retrovectors were produced based on methods described by Burns et al., 1993 (7). A master cell bank of the GPEx–CHO cell line was the parent cell line for all data outlined in this article. Cell lines expressing recombinant proteins and antibodies were produced as shown in Figures 2 and 3. Retrovector transductions were performed at a multiplicity of infection of at least 1000 retrovector particles per CHO cell. To generate recombinant protein expressing cell lines, the parent GPEx–CHO cells were transduced three times with the genetically engineered retrovector. For generation of antibody producing cell lines, an initial transduction of GPEx–CHO cells was performed using a retrovector containing the light chain gene. The light chain containing pool of cells was then transduced with a retrovector containing the heavy chain gene. Upon completion of both transductions, the resulting pool of cells was then transduced a second time with light chain retrovector and two additional times with heavy chain retrovector for a total of five transduction cycles.


Figure 3
Single cell clones were isolated from the final recombinant protein or antibody producing cell pool using limited dilution cloning. Approximately 300–500 clonal lines were screened for protein production levels and various protein specific characteristics. The top 20 clones were selected based on protein production in 96-well plates. Fed-batch (generic conditions) protein production and specific productivity results from triplicate T150-flasks were then used to narrow the number of clones to the top 3–5 candidates. These clones were subsequently moved into shake flask upstream process development for production analysis using a matrix of different fed-batch culture conditions to select the master cell bank candidate clone and the upstream process base conditions for further development. Antibody producing candidate clones in these generic fed-batch conditions typically produce titers of 1–2 g/L and have specific productivities of 25–70 picograms/cell/day (p/c/d). As shown in Table I, after the initial upstream development screening the titers improve to a range from 2–5 g/L, and specific productivities of 40–100 p/c/d are achieved.


Figure 4
To analyze genetic stability, DNA or RNA was isolated from cells in log growth phase. A quantitative real-time PCR based assay was used to estimate the number of gene copies inserted in the cell lines. A DNA sequence present in each transgene insert was used as the target sequence to estimate the total number of insertions. The β 1,4 galactosyltransferase-1 gene is used as an endogenous marker gene to control for the amount of genomic DNA in each reaction. The gene index is calculated by subtracting the transgene assay threshold cycle from the control assay threshold cycle. A similar quantitative real-time PCR assay along with a reverse transcription step was used to determine the level of heavy chain and light chain mRNA being expressed. Either a portion of the constant region of the heavy chain or light chain was used to determine heavy and light chain mRNA levels respectively. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a control mRNA for the CHO cell lines. Isolated RNA was reverse transcribed and three quantitative real-time PCR (heavy chain, light chain, GAPDH control) reactions were run in triplicate. Similar to the assays performed with genomic DNA, a transgene mRNA index value was calculated by subtracting the sample threshold cycle number for the either the heavy chain or light chain assay from the control GAPDH assay threshold cycle value.


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