Per.C6 cells

PER.C6 cells have been used extensively for the production of gene therapy vectors. This cell line was derived from the immortalization of human embryonic retina cells through the use of adenovirus El gene (Fallaux et al., 1998). This cell line has been well characterized since its establishment, and no retroviruses or adventitious viruses have been detected in it. This cell line is easily adapted to different growth conditions and stably produces high levels of recombinant proteins. 

The preferential utilization of glutamine as an energy source is cell-dependent. Maranga and Goochee (2006) have demonstrated that PER.C6 cells fall into a minor category of mammalian cell lines for which glutamine plays a minor role in energy metabolism. 

Maranga and Goochee (2006) have reported that, when PER.C6 cells were cultured in suspension in a 2-liter serum-free bioreactor at a con-... 

Maranga L, Goochee CF (2006), Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels, Biotechnol. Bioeng. 94 139-150. 

The investigations described in this chapter provide an overview of clone generation, fed-batch and perfusion process development, as weU as detailing the history of the PER.C6 cell line, and how it has been characterized. These studies have... 

The DNA construct pIG.ElA.ElB (Fig. 3.2) was used for making PER.C6 cells. The El genes are driven by the human phosphogly-cerate kinase (PGK) promoter, which is a known house-keeping promoter [9] and the poly(A) sequences are derived from the Hepatitis B surface antigen gene [10,11]. 

Fig. 3.2 DNA construct used to generate PER.C6 cells. In this construct, the ElA gene is driven by the human phosphoglycerate kinase (PGK) promoter. Transcription is terminated by the hepatitis B virus surface antigen poly(A) sequences.

Primary HER cells have a limited life span, and can be cultured only a few passages, after which the cells senesce. Transfection of HER cells with El constracts results in transformation and immortalization of the cells, reflected by focus formation in the cultures. This is easily recognized by both macroscopic and microscopic examination of the cultures. Such foci can be isolated and cultured further. In this way, PER.C6 cells were isolated after transfection of primary HER cells with pIG.ElA.ElB [15, 16], The cells were apparently immortalized also, without passing through a crisis phase. 

PER.C6 cells stably express the El proteins. In particular, the 21K and 55K El proteins that counteract apoptosis and p53-mediated cell cycle arrest, respectively are expressed to high levels as compared to, for example, HEK293 cells [15]. We assume that this makes the cells relatively insensitive to apoptosis, and may be one of the fac-... 

Selected PER.C6 cell lines possess low copy numbers, typically below 10 copies per cell as measured by Southern analysis (Fig. 3.6). 

A generic fed-batch process has been developed for the production of monoclonal antibodies in PER.C6 cells. The process typically results in a 3- to 4-fold increase in antibody yields compared to the batch process, with yields of 1-3.5 g L after 16-18 days. The feed strategy is based on the metabolic requirements of the PER.C6 cell line. Metabolic characterization of several antibody-producing cell lines identified nutrients and medium components which... 

Fig. 3.14 (A) Cell and (B) antibody profiles for batch (open symbols) and fed-batch cultures for a PER.C6 cell-line expressing antibody C. The data represent an average of 18 shake flask runs.

Fig. 3.15 Nutrient profile during a fed-batch culture of a PER.C6 cell-line expressing antibody A. The data show the concentration of (A) glutamine (closed triangles), glucose (closed squares) (B) leucine (open squares), cystine (closed circles) and (C) serine (open triangles).

Table 3.2 Summary of results obtained with four PER.C6 cell lines expressing different IgGl...

The first feature investigated was the impact of cell perfusion rate (CSPR) on cell culture performance using a spinfilter as the cell retention device. Fig. 3.17 shows the viable cell concentration of a PER.C6 cell line expressing antibody A operated under the same conditions with two different CSPR implemented at the 7-L scale. 

Fig. 3.17 Cell concentration (xlO cells mL ) versus culture time (days) for PER.C6 cell line expressing antibody A operated at two different cell perfusion rates (CSPR).

---