江苏德芳医药科研有限公司
   
   
 
德芳公司技术推介:环胞素A(Cyclosporin A)

Cyclosporin A  

  Technology  Information  

Yung Shin Pharm. Ind. Co., Ltd

Date: July. 07, 2008  

1. Technology Background and information

Panlabs started Cyclosporin A project from March 1995.

The starting strain was Tolypocladium niveum (= inflatum) UAMH4594. It produced 0.175g /L Cyclosporin A in the original medium. Panlabs used many kinds of mutagens and special screen methods along with medium development to improve yield.  

The strain Tolypocladium inflatum from PF21-1 to PF21-13 have been improved by chemical reagent and UV light treatments, they are not genetically modified organismG.M.O. They are biosafety level 1 microrganism.  

YSP transfer the Cyclosporin A technology focus on fermentation from Panlabs during Nov.1996 ~ June.2000.  

(1)List below is the improvement of Cyclosporin A titer and Cyclosporin A ratio in shake flask (panlabs).  

Strain no.

Titer

(g/L)

Ratio

(%)

Date

UAMH4594

0.175

---

Mar.1995

PF21-1

3.88±0.12

78

Nov.1996

PF21-2

4.51±0.10

72

June.1997

PF21-3

5.08±0.31

71

Sept.1997

PF21-4

5.62±0.21

80

Dec.1997

PF21-5

6.33±0.20

91

Mar.1998

PF21-6

6.93±0.52

89

June.1998

PF21-7

7.63±0.27

90

Sept.1998

PF21-8

10.15±0.39

90

Mar.1999

PF21-9

10.95±0.41

90

June.1999

PF21-10

12.00±0.50

90

Sept.1999

PF21-11

13.4

91

Mar.2000

PF21-12

13.1

95

June.2000

PF21-13

15.2

92

June.2000

 

Fig 1. Genealogy

  Fig 2. Yield improvement of Cyclosporin A in Shake flask  

 (2) List below is the evaluation of Cyclosporin A titer and Cyclosporin A ratio in lab scale (YSP).  

Strain no.

Flask

(g/L)

Cyc A ratio

(%)

30L -Fermentor

(g/L)

Cyc A ratio

(%)

PF21-1

4.0

78

 

 

PF21-2

4.6

72

 

 

PF21-3

5.1

71

 

 

PF21-4

5.6

80

 

 

PF21-5

6.3

91

 

 

PF21-6

7.1

89

6.0

80

PF21-7

7.6

90

7.5

85

PF21-8

10.3

90

10.3

85

PF21-9

11.0

90

12.5

85

PF21-10

11.8

90

 

 

PF21-11

13.5

91

 

 

PF21-12

13.1

95

 

 

#PF21-13

14.5

92

15.0

85

  Titer base on fininal volume  
Yield:± 0.5 g /LCyc A ratio:±2 %  
Flask 8 days  
30L -Fermentor10±1days; initial volume 16 L finial volume about 20L

 (3)CYAy-AR01 5Ton-Fermenter SummaryStrain no. PF21-13  

Batch no.

Harvest titer base on Finial volume ( g/L )

Initial volume (L)

Finial volume (L)

Titer base on Initial volume ( g/L )

Cya ratio (%)

Total ()/Batch

AR01-1 031408-5TC

18.55

2500

3800

28.20

84.42

70.51

AR01-2 032808-5TC

 

14.86

2500

4200

24.97

84.96

62.43

AR01-3 041108-5TC

19.27

2500

4200

32.37

87.83

80.92

Average

17.56

2500

4067

28.51

85.74

71.29 /batch

(213.86 /3batch)

    Turbine impellerwith six blades);impeller diameter 72cm

Temperature at 25 ± 1

Control DO value at above 20 % via adjustment of agitation speed, air-flow, and pressure.

feeding 15(NH4)2SO4 control NH4+ conc. 100~200ppm

start feeding 50sucrose when residual sugarR.S)<4.0control residual sugarR.S)≒2.0%.

feeding NaOH sol’n control pH 4.0±0.2

Harvest at fermentation 240 ±24hrs

2The major cost drivers for Cyclosporin A Fermentation Technology

(1)Strain is the best high Cyclosporin A titer and high Cyclosporin A ratio (%).

(2)Strain is very stable

(2)-1 Frozen stocks ( put 1~2ml of the seed growth in a cryovial ) are stable at -75 for at least 30 weeks.

(2)-2When the cultures are prepared as lyophiles. The lyophiles are quite stable with respect to productivity during lyophilization process.

(3)Fermentation Process is easy to scale up and control

(3)-15Ton - Fermentation titer 30L - Fermentation titer

(3)-2Strain is not sensitive for CO2 and Tank pressure

30L Fermentation can control tank pressure 2.0 atm

5Ton - Fermentation can control tank pressure 1.5 atm

(3)-3Strain is not sensitive for DO Value (Dissolved Oxygen)

30L ; 5Ton Fermentation can keep DO Value0 for a long time and the titer is

also good.