New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode 8th Annual European BioInnovation Leaders Summit 10th - 11th February 2015 Radisson Blu Edwardian Hotel, London Bixente MARTIRENE, MSc Senior Product Manager Asahi Kasei Bioprocess Europe

1. New Hollow Fiber Membranes for
AEX and CEX in Flow-Through Mode
Bixente MARTIRENE, MSc
Senior Product Manager
Asahi Kasei Bioprocess Europe
b.martirene@akbio.eu
8th Annual European BioInnovation Leaders Summit
10th - 11th February 2015
Radisson Blu Edwardian Hotel, London
www.ak-bio.com

2. 1) Pathogen Removal Filters
Content
Introduction
1) Hollow Fiber Membrane for AEX: QyuSpeed D
2) Hollow Fiber Membrane Prototype for CEX
3) Case study:
Combination of CEX Prototype & AEX QyuSpeed D
Conclusion

3. Foundation: 1931; HQ: Tokyo; Employees: 25 000; Turnover: 16 B$
Asahi Kasei Group
3

4. PlanovaTM
Virus removal filters
Asahi Kasei Bioprocess
4

5. PlanovaTM
Virus removal filters
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
Asahi Kasei Bioprocess
5

6. PlanovaTM
Virus removal filters
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
Systems & Equipment
Asahi Kasei Bioprocess
6

7. PlanovaTM
Virus removal filters
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
Systems & Equipment
CellufineTM
Cellulose Beads
Chromatography media
(made by JMC Corporation)
Asahi Kasei Bioprocess
7

8. 1) Pathogen Removal Filters
Introduction

9. 9
Why Membrane vs. Resin ?
ü Mass transfer: “fast” convection vs. “slow” diffusion
ü 5-13 BV*/min vs. 0.5-2 BV/min, low pressure drop
ü 10-20 x higher loading capacity ➔ smaller BV required
ü Easy setup & operation ≈ Dead-End Filter
Introduction
* : Bed Volume = Column Volume = Membrane Volume

10. 10
Introduction
Feedback from our customers:
ü Flow-Through mode preferred for ease of use
ü AEX adsorbers more & more implemented in DSP
ü CEX mainly performed with classical resins

11. 11
Introduction
Asahi Kasei BioProcess:
ü QyuSpeed D launched in 2011 for AEX ➔ Success
ü New Hollow Fiber Membrane for CEX in FT mode ???

12. 1) Pathogen Removal Filters
1) Hollow Fiber Membrane for AEX: QyuSpeed D

13. 13
QyuSpeed D AEX Adsorber
Inlet
Outlet
QyuSpeed D
Module

14. 14
QyuSpeed D AEX Adsorber
Inlet
Outlet
Hollow fiberQyuSpeed D
Module
Protein solution
with biomolecules -
Protein solution
biomolecules - free
§ Dead-End
filtration
§ Removal of
HCP, DNA,
Virus

15. 15
QyuSpeed D AEX Adsorber
Inside the pore
Grafted chain with
DEA ligands
Biomolecules -
Micropore
0.2-0.3 µm
>

16. 16
QyuSpeed D AEX Adsorber
Inside the pore
Grafted chain with
DEA ligands
Biomolecules -
Micropore
0.2-0.3 µm
H2C
C-C-O-CH2CHCH2H3C-
=
O HO
NH(CH2CH3)2
+
()
H2C
C-C-O-CH2CHCH2H3C-
=
O HO
()
NH(CH2CH3)2
+
Poly glycidyl methacrylate
Grafted chain
DEA
> >

17. 17
QyuSpeed D AEX Adsorber
ü Multipoint adsorption
ü High ligand density
Inside the pore
Grafted chain with
DEA ligands
Biomolecules -
Micropore
0.2-0.3 µm
H2C
C-C-O-CH2CHCH2H3C-
=
O HO
NH(CH2CH3)2
+
()
H2C
C-C-O-CH2CHCH2H3C-
=
O HO
()
NH(CH2CH3)2
+
Poly glycidyl methacrylate
Grafted chain
DEA
➔ high DBC & Salt tolerance
> >

18. 18
Product Line-Up
Membrane
(Bed)
Volume
0.6 mL 150 mL 550 mL 5 L
Maximum
Flow Rate
8 mL/min 2 L/min 8 L/min 60 L/min
0.6 mL 150 mL 550 mL 5 L
1172 mm
315 mm

19. 19
Innovative Implementation: pre Prot. A

20. 1) Post Protein A, in place of traditional AEX resin column
2) Post CEX w/o any prior dilution or buffer change: salt tolerant
Protein A
UFPlanova
CIEX
Centri-
fugation
Tangential MF
Depth
Filtration
or
0.2 µm
filtration
20
AEX with
QyuSpeed D
Classical Implementations of QyuSpeed D

21. 10% BSA DBC > 40 g/L-BV
10% DNA DBC @ 15 mS/cm < 30 g/L-BV
HCP reduction
25 – 99 % removal
(depending on pI of HCP)
Parvovirus LRV > 4 Log
MAb Loading capacity
Post Protein A
(in standard flow through mode)
> 2000 g/L-BV
(depending on quantities of impurities)
Number of regenerations
> 10
(tested up to 100 x with BSA)
21
Performances

22. 22
Advantages vs. other AEX Adsorbers
ü Same membrane for low or high conductivity solutions
ü “flexible” implementation: pre or post Prot. A, pre or post CEX
ü Single-use or Reusable (1M NaCl; 1N NaOH)

23. 23
2) Hollow Fiber Membrane Prototype for CEX

24. 24
2) Hollow Fiber Membrane Prototype for CEX
➔ CEX in FT mode

25. 25
New QyuSpeed Prototype for CEX
Inlet
Lab module
Outlet

26. 26
New QyuSpeed Prototype for CEX
Inlet
§ Flow Through
mode = Dead-
End filtration
§ Removal of
Aggregates,
Prot. A, HCP
Outlet
Lab module

27. 27
New QyuSpeed Prototype for CEX
Inlet
Hollow fiber
Protein solution
with aggregates
Protein solution
aggregate free
Outlet
Lab module
§ Flow Through
mode = Dead-
End filtration
§ Removal of
Aggregates,
Prot. A, HCP

28. 28
Inside the pore
Grafted chain with
3 different ligands
Aggregate
Micropore
0.2-0.3 µm
>
New QyuSpeed Prototype for CEX

29. 29
Inside the pore
Grafted chain with
3 different ligands
Aggregate
Micropore
0.2-0.3 µm
Grafted chain
> >
New QyuSpeed Prototype for CEX
-R1
-R2
-R3
R1, R2 & R3 are side chains with different ligands & interactions

30. 30
Aggregate Removal vs. pH

31. 31
Aggregate Removal vs. pH
Feed solution: MAb (h-IgG1), 5 mg/mL, pI 8.4
Buffer: 15 mM Tris-HCl
Conductivity: 1.4 mS/cm
Loading: ~ 1 000 g/L-membrane volume (BV)
Flow rate: 6 BV/min
Aggregate content: ~ 5 %

32. 32
Aggregate Removal vs. pH
Feed solution: MAb (h-IgG1), 5 mg/mL, pI 8.4
Buffer: 15 mM Tris-HCl
Conductivity: 1.4 mS/cm
Loading: ~ 1 000 g/L-membrane volume (BV)
Flow rate: 6 BV/min
Aggregate content: ~ 5 %
pH 7.0 pH 7.5 pH 8.0
Feed
solution
Monomer (%) 94.73 94.51 93.54
Dimer (%) 2.25 2.76 3.58
≧ Trimer (%) 3.02 2.73 2.88

33. 33
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0

34. 34
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Monomer recovery (%) 88.87 92.2 93.07

35. 35
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Monomer recovery (%) 88.87 92.2 93.07
Flow Through
(reduction %)
Monomer (%) 97.42
Dimer (%) 1.34 (49)
≧ Trimer (%) 1.24 (65)

36. 36
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Monomer recovery (%) 88.87 92.2 93.07
Flow Through
(reduction %)
Monomer (%) 97.42 98.65
Dimer (%) 1.34 (49) 1.10 (65)
≧ Trimer (%) 1.24 (65) 0.25 (92)

37. 37
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Monomer recovery (%) 88.87 92.2 93.07
Flow Through
(reduction %)
Monomer (%) 97.42 98.65 97.98
Dimer (%) 1.34 (49) 1.10 (65) 1.74 (57)
≧ Trimer (%) 1.24 (65) 0.25 (92) 0.28 (91)

38. 38
Aggregate Removal vs. pH
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Monomer recovery (%) 88.87 92.2 93.07
Flow Through
(reduction %)
Monomer (%) 97.42 98.65 97.98
Dimer (%) 1.34 (49) 1.10 (65) 1.74 (57)
≧ Trimer (%) 1.24 (65) 0.25 (92) 0.28 (91)

39. 39
Aggregate Removal vs. Cond.

40. 40
Aggregate Removal vs. Cond.
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm

41. 41
Conductivity (mS/cm) 1.4 2.6 4.7
Monomer recovery (%) 88.87 92.32 89.12
Flow Through
(reduction %)
Monomer (%) 97.42 99.48 98.59
Dimer (%) 1.34 (49) 0.39 (78) 1.03 (60)
≧ Trimer (%) 1.24 (65) 0.13 (95) 0.38 (86)
Aggregate Removal vs. Cond.
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm
pH 7.0

42. 42
Comments on the Performances
ü Performances linked to pH & Cond. ➔ optimizations
ü ~ 90 % monomer recovery (binding at the beginning)
ü Up to 95 % aggregate removal possible
ü > 1 000 g/L-BV loading capacity
➔ 10-20 x higher loading capacity vs. traditional CEX resin

43. 1) Pathogen Removal Filters
3) Case study:
Combination of CEX Prototype & AEX QyuSpeed D

44. 44
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL

45. 45
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Elution buffer
25mM Acetate; pH 3.4

46. 46
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

47. 47
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

48. 48
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-BV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

49. 49
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-BV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

50. 50
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-BV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4156 mg/mL-BV
MAb: 2.08 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

51. 51
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype
(FT)

52. 52
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype
(FT)
Monomer 97.6 %
Dimer / ≧Trimer 1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX

53. 53
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype
(FT)
Monomer 97.6 %
Dimer / ≧Trimer 1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
- 88 %
- 20 %
- 83 %
Monomer 99.66 %
Dimer / ≧Trimer 0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %

54. 54
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype
(FT)
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Monomer 97.6 %
Dimer / ≧Trimer 1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
- 88 %
- 20 %
- 83 %
Monomer 99.66 %
Dimer / ≧Trimer 0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %

55. 55
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype
(FT)
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Monomer 97.6 %
Dimer / ≧Trimer 1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
Flow Through of AEX
- 88 %
- 20 %
- 83 %
Monomer 99.66 %
Dimer / ≧Trimer 0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %
Monomer 99.75 %
Dimer / ≧Trimer 0.18 % / < DL
HCP < 1 ppm
Protein A 0.1 ppm
Monomer Recovery: 99 %
- 35 %
- 99 %
- 83 %

56. 1) Pathogen Removal Filters
Conclusion

57. 57
Conclusion
ü Increasing use of membranes in DSP & Flow Through mode preferred

58. 58
Conclusion
ü Increasing use of membranes in DSP & Flow Through mode preferred
ü QyuSpeed D for AEX:
• Unique grafted-chain & hollow fiber design
• Excellent binding capacity & salt tolerance for DNA, HCP, Virus removal
• Very flexible implementation: pre or post Protein A, pre or post CEX
• Single Use or Reusable

59. 59
Conclusion
ü Increasing use of membranes in DSP & Flow Through mode preferred
ü QyuSpeed D for AEX:
• Unique grafted-chain & hollow fiber design
• Excellent binding capacity & salt tolerance for DNA, HCP, Virus removal
• Very flexible implementation: pre or post Protein A, pre or post CEX
• Single Use or Reusable
ü QyuSpeed Prototype for CEX:
• Unique grafted-chain & hollow fiber design
• Flow Through mode
• High aggregate removal & monomer recovery @ > 1000 g/L loading
• Single Use or Reusable

60. 60
Conclusion
ü Increasing use of membranes in DSP & Flow Through mode preferred
ü QyuSpeed D for AEX:
• Unique grafted-chain & hollow fiber design
• Excellent binding capacity & salt tolerance for DNA, HCP, Virus removal
• Very flexible implementation: pre or post Protein A, pre or post CEX
• Single Use or Reusable
ü QyuSpeed Prototype for CEX:
• Unique grafted-chain & hollow fiber design
• Flow Through mode
• High aggregate removal & monomer recovery @ > 1000 g/L loading
• Single Use or Reusable
ü Excellent synergy of both QyuSpeed membranes !

61. 61
Thank you to my Japanese colleagues !
Taniguchi-san
Koguma-san
Shirataki-san
Acknowledgements

62. 62
Thank you to my Japanese colleagues !
Taniguchi-san
Koguma-san
Shirataki-san
And my European Colleagues here today !
Mathithas-san and Konstantin-san
Acknowledgements

63. 63
18th Planova Workshop
ü 22nd & 23rd October 2015 in Athens, Greece
ü Technical presentations by our customers about their experience
with Planova virus removal filters, BioOptimal TFF, QyuSpeed D
ü Social activities: discover of the ancient city & special
entertainments in the evenings
ü Number of attendees: 200
ü YOU ARE WELCOME TO REGISTER !!
ü www.ak-bio.com/planova-workshop/workshop-2015-athens/description/

64. 64
どうもありがとう
Domo Arigato!
(thank you very much)

65. 65
1.3 m1.4 m
Industrial References
ü 2 customers using already QyuSpeed D modules 5L

66. § Impurities removal by AEX (DNA, HCP),
§ Potential enhancement of Prot. A (very expensive step): better
efficiency, easier regeneration, more cycles.
66
Protein A
UFPlanova
AIEX
CIEX
Centri-
fugation
Tangential MF
Depth
Filtration
or
0.2 µm
filtration
AEX with
QyuSpeed D
or much
reduced size
Innovative Implementation: pre Prot. A

67. Protein A
UFPlanova
AIEX
CIEX
Centri-
fugation
Tangential MF
Depth
Filtration
or
0.2 µm
filtration
67
AEX with
QyuSpeed D
or much
reduced size
Other (very) Innovative Implementation:
cell culture harvesting/clarification

68. Protein A
UFPlanova
AIEX
CIEX
Centri-
fugation
Tangential MF
Depth
Filtration
or
0.2 µm
filtration
3 actions in 1 single step:
ü Cell culture clarification (0.2 µm; TFF mode),
ü AEX chromatography (DNA, Virus & HCP removal),
ü “Protection” of Protein A.
68
AEX with
QyuSpeed D
or much
reduced size
Other (very) Innovative Implementation:
cell culture harvesting/clarification

69. 69
Aggregate Removal vs. Cond.

70. 70
Feed solution: MAb (h-IgG1), 5 mg/mL, pI 8.4
Buffer: 15 mM Tris-HCl
pH: 7.0
Loading: ~ 1000 mg/mL-membrane volume (BV)
Flow rate: 6 BV/min
Aggregate Removal vs. Cond.

71. 71
Feed solution: MAb (h-IgG1), 5 mg/mL, pI 8.4
Buffer: 15 mM Tris-HCl
pH: 7.0
Loading: ~ 1000 mg/mL-membrane volume (BV)
Flow rate: 6 BV/min
Conductivity (mS/cm) 1.4 2.6 4.7
Feed
solution
Monomer (%) 94.73 95.94 95.49
Dimer (%) 2.25 1.59 2.2
≧ Trimer (%) 3.02 2.47 2.31
Aggregate Removal vs. Cond.

72. 72
Aggregate Removal vs. Cond.
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm

73. 73
Conductivity (mS/cm) 1.4 2.6 4.7
Monomer recovery (%) 88.87 92.32 89.12
Flow Through
(reduction %)
Monomer (%) 97.42 99.48 98.59
Dimer (%) 1.34 (49) 0.39 (78) 1.03 (60)
≧ Trimer (%) 1.24 (65) 0.13 (95) 0.38 (86)
Aggregate Removal vs. Cond.
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm
pH 7.0

74. 74
Conductivity (mS/cm) 1.4 2.6 4.7
Monomer recovery (%) 88.87 92.32 89.12
Flow Through
(reduction %)
Monomer (%) 97.42 99.48 98.59
Dimer (%) 1.34 (49) 0.39 (78) 1.03 (60)
≧ Trimer (%) 1.24 (65) 0.13 (95) 0.38 (86)
Aggregate Removal vs. Cond.
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm

75. 75
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL

76. 76
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Elution buffer
0.1M Citrate; pH 3.4

77. 77
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

78. 78
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

79. 79
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

80. 80
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1067 mg/mL-MV
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

81. 81
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1067 mg/mL-MV
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4713 mg/mL-MV
MAb: 3.80 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

82. 82
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1067 mg/mL-MV
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4713 mg/mL-MV
MAb: 3.80 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.77 %
Dimer /
≧Trimer
1.32 % / 0.91 %
HCP 390 ppm
Protein A 0.5 ppm
Loading to CEX CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer
0.1M Citrate; pH 3.4

83. 83
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1067 mg/mL-MV
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4713 mg/mL-MV
MAb: 3.80 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.77 %
Dimer /
≧Trimer
1.32 % / 0.91 %
HCP 390 ppm
Protein A 0.5 ppm
Loading to CEX
Flow Through of CEX
- 79 %
- 35 %
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
- 80 %
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Monomer 99.54 %
Dimer /
≧Trimer
0.33 % / 0.14 %
HCP 254 ppm
Protein A 0.1 ppm
Monomer Recovery: 87 %
Elution buffer
0.1M Citrate; pH 3.4

84. 84
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1067 mg/mL-MV
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4713 mg/mL-MV
MAb: 3.80 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.77 %
Dimer /
≧Trimer
1.32 % / 0.91 %
HCP 390 ppm
Protein A 0.5 ppm
Loading to CEX
Flow Through of CEX
Flow Through of AEX
- 79 %
- 35 %
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
- 80 %
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Monomer 99.54 %
Dimer /
≧Trimer
0.33 % / 0.14 %
HCP 254 ppm
Protein A 0.1 ppm
Monomer Recovery: 87 %
Monomer 99.79 %
Dimer /
≧Trimer
0.21 % / < DL
HCP 6 ppm
Protein A < 0.1 ppm
Monomer Recovery: 99 %
- 55 %
- 98 %
Elution buffer
0.1M Citrate; pH 3.4

85. 85
Combination CEX Prototype & AEX QyuSpeed D
0
0.2
0.4
0.6
0.8
1
1.2
0.00 200.00 400.00 600.00 800.00 1000.00
C/C0
Load IgG(mg/mL-ad)
CEX
Monomer
Dimer
≧Trimer
HCP
protein A
0
0.2
0.4
0.6
0.8
1
1.2
0 1000 2000 3000 4000 5000
C/C0
Load IgG(mg/mL-ad)
AEX
Monomer
Dimer
≧Trimer
HCP
Monomer 97.77 %
Dimer /
≧Trimer
1.32 % / 0.91 %
HCP 390 ppm
Protein A 0.5 ppm
Loading to CEX
Flow Through of CEX
Flow Through of AEX
- 79 %
- 35 %
- 80 %
Monomer 99.54 %
Dimer /
≧Trimer
0.33 % / 0.14 %
HCP 254 ppm
Protein A 0.1 ppm
Monomer Recovery: 87 %
Monomer 99.79 %
Dimer /
≧Trimer
0.21 % / < DL
HCP 6 ppm
Protein A < 0.1 ppm
Monomer Recovery: 99 %
- 55 %
- 98 %

86. 86
Combination CEX Prototype & AEX QyuSpeed D
Monomer 97.6 %
Dimer /
≧Trimer
1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
Flow Through of AEX
- 88 %
- 20 %
- 83 %
Monomer 99.66 %
Dimer /
≧Trimer
0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %
Monomer 99.75 %
Dimer /
≧Trimer
0.18 % / < DL
HCP < 1 ppm
Protein A 0.1 ppm
Monomer Recovery: 99 %
- 35 %
- 99 %
- 83 %
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000
C/C0
Load IgG(mg/mL-ad)
CEX (NCX)
Monomer
Dimer
>Trimer
HCP
Protein A
0
0.2
0.4
0.6
0.8
1
1.2
0 1000 2000 3000 4000
C/C0
Load IgG(mg/mL-ad)
AEX (QSD)
Monomer
Dimer
HCP
Protein A

87. 87
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-MV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4156 mg/mL-MV
MAb: 2.08 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.6 %
Dimer /
≧Trimer
1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

88. 88
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-MV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4156 mg/mL-MV
MAb: 2.08 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.6 %
Dimer /
≧Trimer
1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
- 88 %
- 20 %
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
- 83 %
Monomer 99.66 %
Dimer /
≧Trimer
0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %
Titration
pH 7.0
Elution buffer
25mM Acetate; pH 3.4

89. 89
Combination CEX Prototype & AEX QyuSpeed D
Protein A
(BE)
CEX prototype
(FT)
Loading: 1041 mg/mL-MV
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Loading: 4156 mg/mL-MV
MAb: 2.08 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
Monomer 97.6 %
Dimer /
≧Trimer
1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX
Flow Through of CEX
Flow Through of AEX
- 88 %
- 20 %
CHO culture
MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
- 83 %
Monomer 99.66 %
Dimer /
≧Trimer
0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer Recovery: 95 %
Monomer 99.75 %
Dimer /
≧Trimer
0.18 % / < DL
HCP < 1 ppm
Protein A 0.1 ppm
Monomer Recovery: 99 %
- 35 %
- 99 %
Titration
pH 7.0
- 83 %
Elution buffer
25mM Acetate; pH 3.4