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 [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


QyuSpeedTM D AEX adsorber

BioOptimalTM MF-SL TFF microfilter


5




Systems & Equipment


6




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

13

QyuSpeed D AEX Adsorber

Inlet

Outlet

QyuSpeed D Module

14


Inlet

Outlet

Hollow fiber QyuSpeed 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


Biomolecules -


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


ü  Multipoint adsorption ü  High ligand density

Inside the pore


Biomolecules -


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


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


>


29

Inside the pore

Grafted chain with 3 different ligands

Aggregate


Grafted chain

> >


-R1

-R2

-R3

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

30

Aggregate Removal vs. pH

31


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


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


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


Flow Through

(reduction %)

Monomer (%) 97.42

Dimer (%) 1.34 (49)

≧ Trimer (%) 1.24 (65)

36


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


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


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


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

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


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Clarification


Elution buffer 25mM Acetate; pH 3.4

46


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Clarification


Titration pH 7.0


47


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Aggregate addition

Clarification


Titration pH 7.0


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


Titration pH 7.0


49


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)

Aggregate addition

Clarification


Titration pH 7.0


50


Protein A (BE)

CEX prototype (FT)


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


Titration pH 7.0


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


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



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



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


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


AIEX

CIEX

Centri- fugation

Tangential MF

Depth Filtration

or

0.2 µm filtration

67

AEX with QyuSpeed D


Other (very) Innovative Implementation: cell culture harvesting/clarification


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


Other (very) Innovative Implementation: cell culture harvesting/clarification

69


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


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


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


76


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Clarification


Elution buffer 0.1M Citrate; pH 3.4

77


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Clarification


Buffer change 15 mM Tris-HCl pH 7.0; Cond. 1.4 mS/cm


78


Protein A (BE)

CEX prototype (FT)

AEX QyuSpeed D (FT)

Aggregate addition

Clarification




79


Protein A (BE)

CEX prototype (FT)

Titration pH 7.8

AEX QyuSpeed D (FT)

Aggregate addition

Clarification




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




81


Protein A (BE)

CEX prototype (FT)


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




82


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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



83


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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 %


- 80 %


Monomer 99.54 %

Dimer / ≧Trimer

0.33 % / 0.14 %

HCP 254 ppm

Protein A 0.1 ppm



84


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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 %


- 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 %


85


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 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

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


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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


88


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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 %


- 83 %

Monomer 99.66 %

Dimer / ≧Trimer

0.28 % / < DL

HCP 279 ppm

Protein A 0.6 ppm


Titration pH 7.0


89


Protein A (BE)

CEX prototype (FT)


Titration pH 7.8

AEX QyuSpeed D (FT)


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 %


- 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 %


Date post:	15-Apr-2017
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New Hollow Fiber Membranes for AEX and CEX in Flow-Through Mode

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