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
Spermiogenesis or Spermateleosis or metamorphosis of spermatid
New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode
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
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
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
> >
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)
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
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
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/
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
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
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