new hollow fiber membranes for aex and cex in flow-through mode
TRANSCRIPT
New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode
Bixente MARTIRENE, MSc Senior Product Manager Asahi Kasei Bioprocess Europe [email protected]
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 Virus removal filters
QyuSpeedTM D AEX adsorber
BioOptimalTM MF-SL TFF microfilter
Asahi Kasei Bioprocess
5
PlanovaTM Virus removal filters
QyuSpeedTM D AEX adsorber
BioOptimalTM MF-SL TFF microfilter
Systems & Equipment
Asahi Kasei Bioprocess
6
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
1) Pathogen Removal Filters
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) Pathogen Removal Filters 1) Hollow Fiber Membrane for AEX: QyuSpeed D
13
QyuSpeed D AEX Adsorber
Inlet
Outlet
QyuSpeed D Module
14
QyuSpeed D AEX Adsorber
Inlet
Outlet
Hollow fiber QyuSpeed D Module
Protein solution
with biomolecules -
Protein solution
biomolecules - free
§ Dead-End filtration
§ Removal of HCP, DNA, Virus
15
QyuSpeed D AEX Adsorber
Inside the pore
Grafted chain with DEA ligands
Biomolecules -
Micropore 0.2-0.3 µm
>
16
QyuSpeed D AEX Adsorber
Inside the pore
Grafted chain with DEA ligands
Biomolecules -
Micropore 0.2-0.3 µm
H2C
C-C-O-CH2CHCH2 H3C-
=
O HO
NH(CH2CH3)2 +
( )
H2C
C-C-O-CH2CHCH2 H3C-
=
O HO
( )
NH(CH2CH3)2 +
Poly glycidyl methacrylate
Grafted chain
DEA > >
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-CH2CHCH2 H3C-
=
O HO
NH(CH2CH3)2 +
( )
H2C
C-C-O-CH2CHCH2 H3C-
=
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 UF Planova
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
2) Hollow Fiber Membrane Prototype for CEX
24
2) Hollow Fiber Membrane Prototype for CEX
➔ CEX in FT mode
25
New QyuSpeed Prototype for CEX
Inlet
Lab module
Outlet
26
New QyuSpeed Prototype for CEX
Inlet
§ Flow Through mode = Dead-End filtration
§ Removal of Aggregates, Prot. A, HCP
Outlet
Lab module
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
Inside the pore
Grafted chain with 3 different ligands
Aggregate
Micropore 0.2-0.3 µm
>
New QyuSpeed Prototype for CEX
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
Aggregate Removal vs. pH
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
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
Aggregate Removal vs. pH pH 7.0 pH 7.5 pH 8.0
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
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
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
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
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
Aggregate Removal vs. Cond.
40
Aggregate Removal vs. Cond. 1.4 mS/cm 2.6 mS/cm 4.7 mS/cm
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
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
1) Pathogen Removal Filters 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
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
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
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
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
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
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
Combination CEX Prototype & AEX QyuSpeed D
CEX prototype (FT)
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
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
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
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 %
1) Pathogen Removal Filters
Conclusion
57
Conclusion ü Increasing use of membranes in DSP & Flow Through mode preferred
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
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
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
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DomoArigato!
(thankyouverymuch)
65
1.3 m 1.4 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 UF Planova
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 UF Planova
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 UF Planova
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
Aggregate Removal vs. Cond.
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
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
Aggregate Removal vs. Cond. 1.4 mS/cm 2.6 mS/cm 4.7 mS/cm
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
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
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
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
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
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
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
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
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
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 CEXCHO 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
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
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
Combination CEX Prototype & AEX QyuSpeed D
0
0.2
0.4
0.6
0.8
1
1.2
0.00 200.00 400.00 600.00 800.001000.00
C/C0
LoadIgG(mg/mL-ad)
CEX
Monomer
Dimer
≧Trimer
HCP
proteinA
0
0.2
0.4
0.6
0.8
1
1.2
0 1000 2000 3000 4000 5000
C/C0
LoadIgG(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
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
LoadIgG(mg/mL-ad)
CEX(NCX)
Monomer
Dimer
>Trimer
HCP
ProteinA
0
0.2
0.4
0.6
0.8
1
1.2
0 1000 2000 3000 4000
C/C0
LoadIgG(mg/mL-ad)
AEX(QSD)
Monomer
Dimer
HCP
ProteinA
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 CEXCHO culture MAb (h-IgG1), pI 8.4, Titer < 1 mg/mL
Titration pH 7.0
Elution buffer 25mM Acetate; pH 3.4
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
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