Systematic development of temperature shift strategies for Chinese hamster ovary cells based on short duration cultures and kinetic modeling

Figures & data

Table 1. Kinetic parameters for cell growth, protein production and lactate calculated from the 8-day short duration fed-batch study with GS CHO1 cell line.

Temperature	°C	32	33	34	35	36.5
Cell Growth	μmax (hr^−^1)	0.36	0.44	0.52	0.90	0.93
	kd (day^−^1)	0.05	0.14	0.23	0.26	0.49
Protein	KP (g· day^−^1· 10^−^9cells^−^1)	0.48	0.46	0.44	0.40	0.37
Lactate	aLac (g· 10^−^12cells^−^1· hr^−^1)	10	8.1	5.5	4.6	4.1
	bLac (g· 10^−^9cells^−^1)	0.83	1.2	1.6	2.3	5.6
	kLac (g· 10^−^11cells^−^1· hr^−^1)	0.87	1.1	1.1	1.8	2.6

Figure 1. CHO1 cell growth (A), metabolite profiles: glucose (B), glutamine (C), glutamate (D), and lactate (E), and mAb1 production: titer (F) and specific productivity q_P (G) in fed-batch production in 250-mL shake flasks at different constant temperatures of 32, 33, 34, 35, and 36.5 ºC with a high initial VCD of 10 × 10⁶ cells/mL for an 8-day short duration. The dots denote the experimental data with the average of duplicate runs (n = 2) and the lines represent the simulation results.

Table 2. Predicted mAb1 titer (normalized) for different TS conditions via kinetic models from the 8-day short duration fed-batch study, assumed that the GS CHO1 cell cultures would be inoculated at a typical VCD of 0.6 × 10⁶ cells/mL with an initial temperature of 36.5 °C, then downshift to different temperatures for a 14-day full duration. The validated titer (normalized) values were from the GS CHO1 cell cultures described in Figure 3. The 8 conditions with bold produced high titer values ≥97%.

TS strategy	Shift1 day	Shift1 to temperature (°C)	Shift2 day	Shift2 to temperature (°C)	Predicted titer (%)	Validated titer (%)
1	NA				89.1	NA
2	6	32	NA		82.1
3		33			87.9	90.2
4		34			92.3	92.3
5		35			92.4	NA
6	8	32			87.3
7		33			95.1	96.8
8		34			97.8	97.8
9		35			99.0	NA
10	10	32			92.1
11		33			100.0
12		34			98.5
13		35			93.8
14 ^f	6	35	8	32	87.2
15				33	94.9
16				34	97.4
17		34		32	87.3
18				33	94.5	94.2
19		33		32	83.3	NA
20	6	35	10	32	87.7
21				33	95.2
22				34	97.6
23		34		32	92.8
24				33	94.8
25		33		32	87.0
26	8	35	10	32	88.1
27				33	95.4
28				34	97.9
29		34		32	98.1
30				33	95.1	95.1
31		33		32	90.6	NA

Figure 2. CHO1 model validation for VCD (A), titer (B) and specific productivity (C) in fed-batch production 250-mL shake flasks with TS from 36.5 to 33 or 34 °C on different days using a typical initial VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration. The dots denote the experimental data with the average of duplicate runs (n = 2) and the lines represent the computing results using the kinetic models from the 8-day short-duration study.

Figure 3. CHO2 cell growth (A) and metabolite profiles: glucose (B), glutamine (C), glutamate (D), lactate (E) and ammonium (F) in batch production 5-L bioreactors at different constant temperatures of 33, 34, 35, 36.5, 37.5 and 38.5 ºC with an initial typical VCD of 0.6 × 10⁶ cells/mL for a 4-day short duration. The dots denote the experimental data (n = 1) and the lines represent the simulation results.

Figure 4. CHO2 model validation for cell growth (A) and metabolites: glucose (B), glutamine (C), glutamate (D), lactate (E) and ammonium (F) in fed-batch production 5-L bioreactors (n = 3) with TS from 36.5 to 35 °C on day4 using an initial typical VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration. The dots denote the experimental data for a 14-day full duration and the lines represent the computing results for first 10-day duration using the kinetic models from the 4-day short duration study.

Figure 5. VCD profiles prediction of CHO2 at different TS conditions with a typical initial VCD of 0.6 × 10⁶ cells/mL: TS from 36.5 °C to 35, 34 and 33 °C on day 4 (A); TS from 36.5 °C to 33 °C on day 4, day 6 and day 8 (B). M represents a multiple temperature shifting steps: TS from 36.5 °C to 35 °C on day 4, then to 34 °C on day 6, and finally to 33°C on day 8. The lines represent the computational prediction results using the kinetic models from the 4-day short-duration study.

Table 3. Kinetic parameters for cell growth and lactate calculated from the 4-day short duration batch study with DG44 CHO2 cell line.

Temperature	°C	33	34	35	36.5	37.5	38.5
Cell Growth	μmax (hr^−^1)	0.059	0.116	0.121	0.126	0.128	0.120
	kd (day^−^1)	0.048	0.065	0.084	0.13	0.13	0.14
Lactate	aLac (g· 10^−^11cells^−^1· hr^−^1	4.0	5.0	5.0	7.7	8.0	8.1
	bLac (g· 10^−^10cells^−^1)	2.0	1.6	1.4	1.4	1.1	0.76
	kLac (g· 10^−^11cells^−^1· hr^−^1)	2.2	2.8	2.9	4.3	4.4	4.4

Figure 6. Effect of different constant temperatures on CHO2 for mAb2 specific productivity (A) and quality attributes: SEC profile (HMW: B) and charge variants (Acidic: C, Basic: D) in batch 125-mL shake flasks with a high initial VCD of 10 × 10⁶ cells/mL for a 2-day short duration (n = 2).

Figure 7. Effect of different TS conditions on mAb2 specific productivity (q_P values were calculated from day6 to day10) (A) and final D14 quality attributes: SEC profile (HMW: B) and charge variants (Acidic: C, Basic: D) in fed-batch production 5-L bioreactors (n = 2) with an initial temperature of 36.5 °C, then reduced to 35, 33.5 or 32 °C from day5 to day14 using a typical initial VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration.

Table

a	substrate consumption to maintain cell viability
b	chemical generation constant
C	chemical that will generate glutamine or glutamate
CHO	Chinese hamster ovary
d	constant that reflects the overall impact of certain chemical generation and consumption
Glc	glucose
HMW	high molecular weight
k	rate constant
K	saturation number in Michaelis-Menten model
KD	cell death saturation constants
KI	cell growth saturation constants
KP	maximum specific protein production rate
mAb	monoclonal antibody
P	concentration of toxic metabolites
QTP	specific therapeutic protein production rate
r	reaction rate
S	concentration of the limiting substrate
SEC	size-exclusion chromatography
TS	temperature shift
VCD	viable cell density
X	VCD
kd	maximum specific cell death rate
μd	specific death rate
μG	specific growth rate
μmax	maximum specific cell growth rate
V	bulk solution volume
Vi	volume of added nutrient at the ith time
YF	factor of substrate consumption associated with cell growth

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