Supporting Materials

SUPPORTING MATERIALS

SUPPORTING MATERIAL 1:

Supplementary Figure 1: p1010-, p901-EPO plasmid constructs

(A)

(B)

Plasmid constructs used to generate (A) UCOE-containing (B) and non-UCOE-containing CHO-EPO cell lines.

SUPPORTING MATERIAL 2:

Supplementary Figure 2:

(A)(B)

Comparison of growth characteristics between initial and amplified cell lines over Long-term culture.CHO-EPO cell lines were cultured in T-75 flasks using RPMI medium plus 4mM L-Glutamine and 10% (v/v) FBS. Cells were cultured every 2-3 days up to 77 days. Batch growth cultures were created at an initial density of 1x105 cells/mL in 6-well plates, at early (day 0) and late (day 77) generations. Viable cell numbers were measured using light microscopy and trypan blue exclusion method. Values are presented as average of each group ±SEM (n=3). (A) UCOE cell lines, (B) non-UCOE cell lines. Initial cell lines at early generation; Initial cell lines at late generation; Amplified cell lines at early generation; Amplified cell lines at late generation in the presence of MTX; Amplified cell lines at late generation in the absence of MTX.

SUPPORTING MATERIAL 3:

The media samples for volumetric EPO production analyses were taken after 5 days in batch culture for clones shown in Figure 3. However, in Figure 5A the maximum volumetric EPO productions are shown for each cell line. This was analyzed during batch culture on days 2, 3, 4, 5, 7, 8, 9, and 10. The cell lines achieved their maximum EPO production on different days of batch culture (i.e. NE1 on day 7, NE2 on day 10, NE3 on day 8, UE1 on day 7, UE2 on day 8, UE3 on day 7). Therefore, the data in Figure 3 and 5A for top three producing cell lines are not exactly the same. Please see the supplementary Table I for comparison of EPO production at day 5 of batch culture and maximum volumetric production.

Supplementary Table I: Volumetric EPO production in batch culture

Cell lines / Volumetric EPO production mg/L (± SD)
On day 5 of batch growth culture) / Maximum production (days in batch growth culture)
UE1 / 127.4 (±5) / 184.5 (±4.1) on day 7
UE2 / 130.7 (±1) / 196.4 (±7.9) on day 8
UE3 / 74.9 (±0.1) / 138.7 (±9.3) on day 7
NE1 / 64.9 (±1.4) / 137.6 (±11.1) on day 7
NE2 / 41.8 (±4.2) / 67.5 (±1.8) on day 10
NE3 / 33.6 (±5.2) / 52.4 (±3.3) on day 8

Volumetric EPO production was measured for cell lines at early generation during batch growth culture. Samples of culture medium supernatant were taken on days 2, 3, 5, 7, 8, 9 and 10. Volumetric EPO production on day 5 and maximum EPO production achieved for each cell line are shown.