New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode

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

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

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

PlanovaTM
Virus removal filters
Asahi Kasei Bioprocess
4

PlanovaTM
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
5

PlanovaTM
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
Systems & Equipment
6

PlanovaTM
QyuSpeedTM D
AEX adsorber
BioOptimalTM MF-SL
TFF microfilter
Systems & Equipment
CellufineTM
Cellulose Beads
Chromatography media
(made by JMC Corporation)
7

Introduction

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

1)  Hollow Fiber Membrane for AEX: QyuSpeed D

13
QyuSpeed D AEX Adsorber
Inlet
Outlet
QyuSpeed D
Module

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

15
Inside the pore
Grafted chain with
DEA ligands
Biomolecules -
Micropore
0.2-0.3 µm
>

16
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
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
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
Innovative Implementation: pre Prot. A

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

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
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

24
➔ CEX in FT mode

25
New QyuSpeed Prototype for CEX
Inlet
Lab module
Outlet

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

27
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
Inside the pore
Grafted chain with
3 different ligands
Aggregate
Micropore
0.2-0.3 µm
>

29
Inside the pore
Grafted chain with
3 different ligands
Aggregate
Micropore
0.2-0.3 µm
Grafted chain
> >
-R1

-R2
-R3
R1, R2 & R3 are side chains with different ligands & interactions

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
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
pH 7.0 pH 7.5 pH 8.0

34
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
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Flow Through
(reduction %)
Monomer (%) 97.42
Dimer (%) 1.34 (49)
≧ Trimer (%) 1.24 (65)

36
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
Flow Through
(reduction %)
Monomer (%) 97.42 98.65
Dimer (%) 1.34 (49) 1.10 (65)
≧ Trimer (%) 1.24 (65) 0.25 (92)

37
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
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
pH 7.0 pH 7.5 pH 8.0
pH 7.0 pH 7.5 pH 8.0
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
Aggregate Removal vs. Cond.

40
1.4 mS/cm 2.6 mS/cm 4.7 mS/cm

41
Conductivity (mS/cm) 1.4 2.6 4.7
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)
pH 7.0

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

3)  Case study:
Combination of CEX Prototype & AEX QyuSpeed D

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
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
Elution buffer
25mM Acetate; pH 3.4

46
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
Titration
pH 7.0
Elution buffer

47
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
Titration
pH 7.0
Elution buffer

48
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
Titration
pH 7.0
Elution buffer

49
Protein A
(BE)
CEX prototype
(FT)
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
Titration
pH 7.0
Elution buffer

50
Protein A
(BE)
CEX prototype
(FT)
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
MAb: 2.08 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
CHO culture
Titration
pH 7.0
Elution buffer

51
CEX prototype
(FT)

52
CEX prototype
(FT)
Monomer 97.6 %
Dimer / ≧Trimer 1.79 % / 0.56 %
HCP 349 ppm
Protein A 3.6 ppm
Loading to CEX

53
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
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 %
HCP 279 ppm
Protein A 0.6 ppm

55
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 %
HCP 279 ppm
Protein A 0.6 ppm
Monomer 99.75 %
HCP < 1 ppm
Protein A 0.1 ppm
- 35 %
- 99 %
- 83 %

Conclusion

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

58
Conclusion
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
Conclusion
ü  QyuSpeed Prototype for CEX:
•  Flow Through mode
•  High aggregate removal & monomer recovery @ > 1000 g/L loading

60
Conclusion
ü  QyuSpeed Prototype for CEX:
•  Flow Through mode
•  High aggregate removal & monomer recovery @ > 1000 g/L loading
ü  Excellent synergy of both QyuSpeed membranes !

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

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

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

§  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

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

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

70
pH: 7.0
Loading: ~ 1000 mg/mL-membrane volume (BV)
Flow rate: 6 BV/min

71
pH: 7.0
Loading: ~ 1000 mg/mL-membrane volume (BV)
Flow rate: 6 BV/min
Feed
solution
Monomer (%) 94.73 95.94 95.49
Dimer (%) 2.25 1.59 2.2
≧ Trimer (%) 3.02 2.47 2.31

72

73
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)
pH 7.0

74
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)

75
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture

76
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
Elution buffer
0.1M Citrate; pH 3.4

77
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Clarification
CHO culture
Buffer change
15 mM Tris-HCl
pH 7.0; Cond. 1.4 mS/cm
Elution buffer

78
Protein A
(BE)
CEX prototype
(FT)
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
Buffer change
15 mM Tris-HCl
Elution buffer

79
Protein A
(BE)
CEX prototype
(FT)
Titration
pH 7.8
AEX QyuSpeed D
(FT)
Aggregate
addition
Clarification
CHO culture
Buffer change
15 mM Tris-HCl
Elution buffer

80
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
Buffer change
15 mM Tris-HCl
Elution buffer

81
Protein A
(BE)
CEX prototype
(FT)
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
MAb: 3.80 mg/ml
Flow rate: 6 BV/min
Aggregate
addition
Clarification
CHO culture
Buffer change
15 mM Tris-HCl
Elution buffer

82
Protein A
(BE)
CEX prototype
(FT)
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
Buffer change
15 mM Tris-HCl
Elution buffer

83
Protein A
(BE)
CEX prototype
(FT)
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
- 80 %
Buffer change
15 mM Tris-HCl
Monomer 99.54 %
Dimer /
≧Trimer
0.33 % / 0.14 %
HCP 254 ppm
Protein A 0.1 ppm
Elution buffer

84
Protein A
(BE)
CEX prototype
(FT)
MAb: 5.22 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
- 80 %
Buffer change
15 mM Tris-HCl
Monomer 99.54 %
Dimer /
≧Trimer
0.33 % / 0.14 %
HCP 254 ppm
Protein A 0.1 ppm
Monomer 99.79 %
Dimer /
≧Trimer
0.21 % / < DL
HCP 6 ppm
Protein A < 0.1 ppm
- 55 %
- 98 %
Elution buffer

85
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 99.79 %
Dimer /
≧Trimer
0.21 % / < DL
HCP 6 ppm
Protein A < 0.1 ppm
- 55 %
- 98 %

86
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 99.75 %
Dimer /
≧Trimer
0.18 % / < DL
HCP < 1 ppm
Protein A 0.1 ppm
- 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
Protein A
(BE)
CEX prototype
(FT)
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
Titration
pH 7.0
Elution buffer

88
Protein A
(BE)
CEX prototype
(FT)
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
- 83 %
Monomer 99.66 %
Dimer /
≧Trimer
0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Titration
pH 7.0
Elution buffer

89
Protein A
(BE)
CEX prototype
(FT)
MAb: 2.6 mg/mL
Flow rate: 6 BV/min
Titration
pH 7.8
AEX QyuSpeed D
(FT)
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
- 83 %
Monomer 99.66 %
Dimer /
≧Trimer
0.28 % / < DL
HCP 279 ppm
Protein A 0.6 ppm
Monomer 99.75 %
Dimer /
≧Trimer
0.18 % / < DL
HCP < 1 ppm
Protein A 0.1 ppm
- 35 %
- 99 %
Titration
pH 7.0
- 83 %
Elution buffer

New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode

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