1. QyuSpeed D
AEX chromatography membrane,
a highly cost effective technology
5th ANNUAL BIO INNOVATION
15-16th February 2012
London UK
Bixente MARTIRENE – MSc Chem. Eng.
Product Manager
Asahi Kasei Bioprocess Europe
2. Content
PART 1 – Asahi Kasei Bioprocess
PART 2 – QyuSpeed D
PART 3 – Membrane vs. Resin
PART 4 – Performances
PART 5 – Cost simulation: Q resin,
Single Use Q
membrane,
QyuSpeed
D
5. Asahi Kasei Bioprocess
For 20 years, leader in virus removal
filtration with Planova filters range.
From 4.0 m2 down to 0.001 m2 surface area
5
6. Asahi Kasei Bioprocess
For 20 years, leader in virus removal
filtration with Planova filters range.
From 2010, new separation and purification
solutions, for biopharmaceuticals production.
New products based on Asahi’s core
technology (hollow fibers) and high quality
standards.
6
9. Specifications
Filter Format Hollow Fiber
Ligand type Anion exchange
Ligand DEA : -N+H-(CH2CH3)2
Pore Size 0.2 - 0.3 µm
Membrane Material Polyethylene
Fiber ID / OD 2.2 mm / 3.6 mm
Operating Pressure < 2 bar
Reusable after regeneration Yes
Outlet
Inlet
Drain
Hollow fiber 9
10. Pore structure and ligand configuration
Poly (glycidyl methacrylate)
Chain (backbone of the grafted chain)
micropore
)
H3C- C-C-O-CH2CHCH2
=
H 2C O HO N(CH2CH3)2
) (
H3C- C-C-O-CH2CHCH2
=
O HO N(CH2CH3)2
H 2C
(
Micropore
Nominal pore size: Grafted chain Anion-exchange group (DEA)
0.2-0.3 µm
Flow
direction Ligand density: 0.55-0.6 mmol/mL-ads
1200um
SEM x25 10
11. Separation mechanism
Selectivity by ligand type.
Grafted chains Multipoint adsorption
High Dynamic Binding Capacity (DBC).
Protein solution
Proteins - Proteins +
Convective flow
Grafted chain
with Ligands
Hollow Fiber
Pore of the membrane
11
12. Application
Removal of impurities from protein/MAb solutions:
DNA, Viruses, HCP, Prion, Endotoxin.
Host cell protein
Endotoxin
Isoelectric
DNA Viruses
Point
2 7 MAbs 12
Binding of Flow through of
impurities - pH protein/MAb +
buffer
12
13. Product line
Lab-filter
Membrane Volume 0.6 mL
Max Flow rate 8 mL/min
Availability From 2010
2-inch-filter
Membrane Volume 150 mL
Max Flow rate 2 L/min
Availability Mid 2011
4-inch-filter
Membrane Volume 550 mL
Max Flow rate 8 L/min
Availability End 2011
QyuSpeedD-5L end 2012!
315 mm
13
14. Where implementing QyuSpeed D ?
Before or after Protein A.
High salt tolerance = possible after CEX
without any prior dilution.
Centri- AIEX
Depth
fugation Filtration
or
Protein A
0.2 µm Planova UF
filtration
CIEX
Tangential MF
14
15. Where implementing QyuSpeed D ?
After cell culture, 3 actions in 1 single step:
Cell culture clarification,
AEX chromatography,
“Protection” of Protein A.
Centri- AIEX
Depth
fugation Filtration
or
Protein A
0.2 µm Planova UF
filtration
CIEX
Tangential MF
15
17. Membrane vs. Resin
Convective vs. diffusive mass transfer.
DBC independent of the flow rate.
5-20 MV*/min vs. 0.5-2 CV**/min.
Very high flow rates w/o high pressure drop.
*MV: Membrane Volume
**CV: Column Volume
Electron micrograph Electron micrograph
of QyuSpeed D of resin beads
17
18. Membrane vs. Resin
Resin columns can be oversized because :
slow binding mechanism by diffusion,
bed height limitation (~ 20-30 cm) to avoid too
high pressure drop,
poor salt tolerance,
lower impurities DBC than claimed BSA DBC.
18
19. Membrane vs. Resin
To purify the same protein solution:
Membrane Volume (MV) << Column Volume (CV)
MAb loading capacity: often > 2 000 g/L-QSD
19
21. Main AEX membranes - Performances
Q resin Membrane 1 Membrane 2 Membrane 3 QyuSpeed D
Configuration Beads Rolled Sheet Open Pleated Stacked Flat Sheet Hollow fibers
Medium Material Agarose Cellulose PES PE PE
-N+-(CH3)3 -N+-(CH3)3 Quaternary -N+H-
Ligand Primary amine
amine (CH2CH3)2
Max Flow rate 1 CV/min 30 MV/min 10 MV/min 12.5 MV/min 13.5 MV/min
Pore Size - > 3 µm 0.8 µm 0.65 µm 0.2-0.3 µm
10% BSA DBC
> 100 > 29 > 60 > 50 > 40
(g/L-medium)
10% DNA DBC
<5 < 10 < 35 < 35 < 35
(g/L-medium)
at ~ 0 mS/cm
10% DNA DBC < 30
Not applicable.
<2 < 30 < 30
(g/L-medium) (if another Max 5 mS/cm
type)
at 15 mS/cm
Virus LRV > 3-4 > 3-4 > 3-4 >4 >4
Usually much lower reduction than for DNA and Viruses. Very much depending on the operating
HCP reduction
conditions and HCP characteristics (large pI range).
Number of
Up to 100 Single use Not precised Single use > 10
regenerations
21
22. Main AEX membranes - Performances
Q resin Membrane 1 Membrane 2 Membrane 3 QyuSpeed D
Configuration Beads Rolled Sheet Open Pleated Stacked Flat Sheet Hollow fibers
Medium Material Agarose Cellulose PES PE PE
-N+-(CH3)3 -N+-(CH3)3 Quaternary -N+H-
Ligand Primary amine
amine (CH2CH3)2
Max Flow rate 1 CV/min 30 MV/min 10 MV/min 12.5 MV/min 13.5 MV/min
Pore Size - > 3 µm 0.8 µm 0.65 µm 0.2-0.3 µm
10% BSA DBC
> 100 > 29 > 60 > 50 > 40
(g/L-medium)
10% DNA DBC
<5 < 10 < 35 < 35 < 35
(g/L-medium)
at ~ 0 mS/cm
10% DNA DBC < 30
Not applicable.
<2 < 30 < 30
(g/L-medium) (if another Max 5 mS/cm
at 15 mS/cm type)
Virus LRV > 3-4 > 3-4 > 3-4 >4 >4
Usually much lower reduction than for DNA and Viruses. Very much depending on the operating
HCP reduction
conditions and HCP characteristics (large pI range).
Number of
Up to 100 Single use Not precised Single use > 10
regenerations
22
23. Main AEX membranes - Performances
Q resin Membrane 1 Membrane 2 Membrane 3 QyuSpeed D
Configuration Beads Rolled Sheet Open Pleated Stacked Flat Sheet Hollow fibers
Medium Material Agarose Cellulose PES PE PE
-N+-(CH3)3 -N+-(CH3)3 Quaternary -N+H-
Ligand Primary amine
amine (CH2CH3)2
Max Flow rate 1 CV/min 30 MV/min 10 MV/min 12.5 MV/min 13.5 MV/min
Pore Size - > 3 µm 0.8 µm 0.65 µm 0.2-0.3 µm
10% BSA DBC
> 100 > 29 > 60 > 50 > 40
(g/L-medium)
10% DNA DBC
<5 < 10 < 35 < 35 < 35
(g/L-medium)
at ~ 0 mS/cm
10% DNA DBC < 30
Not applicable.
<2 < 30 < 30
(g/L-medium) (if another Max 5 mS/cm
type)
at 15 mS/cm
Virus LRV > 3-4 > 3-4 > 3-4 >4 >4
Usually much lower reduction than for DNA and Viruses. Very much depending on the operating
HCP reduction
conditions and HCP characteristics (large pI range).
Number of
Up to 100 Single use Not precised Single use > 10
regenerations
23
24. Main AEX membranes - Performances
Q resin Membrane 1 Membrane 2 Membrane 3 QyuSpeed D
Configuration Beads Rolled Sheet Open Pleated Stacked Flat Sheet Hollow fibers
Medium Material Agarose Cellulose PES PE PE
-N+-(CH3)3 -N+-(CH3)3 Quaternary -N+H-
Ligand Primary amine
amine (CH2CH3)2
Max Flow rate 1 CV/min 30 MV/min 10 MV/min 12.5 MV/min 13.5 MV/min
Pore Size - > 3 µm 0.8 µm 0.65 µm 0.2-0.3 µm
10% BSA DBC
> 100 > 29 > 60 > 50 > 40
(g/L-medium)
10% DNA DBC
<5 < 10 < 35 < 35 < 35
(g/L-medium)
at ~ 0 mS/cm
10% DNA DBC < 30
Not applicable.
<2 < 30 < 30
(g/L-medium) (if another Max 5 mS/cm
type)
at 15 mS/cm
Virus LRV > 3-4 > 3-4 > 3-4 >4 >4
Usually much lower reduction than for DNA and Viruses. Very much depending on the operating
HCP reduction
conditions and HCP characteristics (large pI range).
Number of
Up to 100 Single use Not precised Single use > 10
regenerations
24
25. Main AEX membranes - Performances
Q resin Membrane 1 Membrane 2 Membrane 3 QyuSpeed D
Configuration Beads Rolled Sheet Open Pleated Stacked Flat Sheet Hollow fibers
Medium Material Agarose Cellulose PES PE PE
-N+-(CH3)3 -N+-(CH3)3 Quaternary -N+H-
Ligand Primary amine
amine (CH2CH3)2
Max Flow rate 1 CV/min 30 MV/min 10 MV/min 12.5 MV/min 13.5 MV/min
Pore Size - > 3 µm 0.8 µm 0.65 µm 0.2-0.3 µm
10% BSA DBC
> 100 > 29 > 60 > 50 > 40
(g/L-medium)
10% DNA DBC
<5 < 10 < 35 < 35 < 35
(g/L-medium)
at ~ 0 mS/cm
10% DNA DBC < 30
Not applicable.
<2 < 30 < 30
(g/L-medium) (if another Max 5 mS/cm
type)
at 15 mS/cm
Virus LRV > 3-4 > 3-4 > 3-4 >4 >4
Usually much lower reduction than for DNA and Viruses. Very much depending on the operating
HCP reduction
conditions and HCP characteristics (large pI range).
> 10
Number of
Up to 100 Single use Not precised Single use
regenerations (up to 100
possible)
25
26. QyuSpeed D advantages
Shorter processing time.
High salt tolerance.
MV << CV (= less chromatographic medium).
Lower buffer consumptions.
26
27. QyuSpeed D advantages
Shorter processing time.
High salt tolerance.
MV << CV (= less chromatographic medium).
Lower buffer consumptions.
No packing.
Less labor costs.
Less hardware equipment.
Smaller footprint.
Easy/reliable scale up by adding modules.
27
29. PART 5 – Cost simulation:
Q resin, Single Use Q membrane
and QyuSpeed D,
for different batch sizes and
different numbers of batches/year
30. Cost simulation - Hypothesis
Comparisons between QyuSpeed D, single use Q membrane, Q resin.
MAb production from CHO cell culture with standard DSP steps.
Each technology leads to similar purification yields.
Protein concentration before AEX: 2 g/L.
Conductivity of the MAb solution before loading: < 5 mS/cm.
3 loading volumes:100 L (small), 1 500 L (medium), 5 000 L (large)
Number of batches : 10/year, 40/year
30
31. Cost simulation - Hypothesis
Comparisons between QyuSpeed D, single use Q membrane, Q resin.
MAb production from CHO cell culture with standard DSP steps.
Each technology leads to similar purification yields.
Protein concentration before AEX: 2 g/L.
Conductivity of the MAb solution before loading: < 5 mS/cm.
3 loading volumes:100 L (small), 1 500 L (medium), 5 000 L (large)
Number of batches : 10/year, 40/year
Cost calculations based on 5 years operation.
Q resin: 100 uses; QyuSpeed D: 15 uses.
Buffer volume for packing step is not counted.
P r i c e s :
- Q resin: 700 $/L-medium;
- Q membrane: 7 000 $/L-
medium; - QyuSpeed D: 14 000 $/L-
medium.
Labor cost: 350 $/h (2 operators) 31
32. Buffer volumes for 1 cycle
Q membrane QyuSpeed D
Process step Q resin
(single use) (reusable)
Flush WFI
(CV* or MV**)
Pre-wash with buffer
(CV or MV)
Equilibration with buffer
(CV or MV)
Washing with buffer
(CV or MV)
Regeneration
with NaCl and NaOH
(CV or MV)
Total buffer volume per cycle
(CV or MV)
*CV: Column Volume
**MV: Membrane Volume
32
33. Buffer volumes for 1 cycle
Q membrane QyuSpeed D
Process step Q resin
(single use) (reusable)
Flush WFI
5 10 -
(CV* or MV**)
Pre-wash with buffer
5 10 10
(CV or MV)
Equilibration with buffer
5 10 15
(CV or MV)
Washing with buffer
5 10 10
(CV or MV)
Regeneration
with NaCl and NaOH 5 - 50
(CV or MV)
Total buffer volume per cycle
(CV or MV) 25 40 85
*CV: Column Volume
**MV: Membrane Volume
33
34. Buffer volumes for 1 cycle
Q membrane QyuSpeed D
Process step Q resin
(single use) (reusable)
Flush WFI
5 10 -
(CV* or MV**)
Pre-wash with buffer
5 10 10
(CV or MV)
Equilibration with buffer
5 10 15
(CV or MV)
Washing with buffer
5 10 10
(CV or MV)
Regeneration
with NaCl and NaOH 5 - 50
(CV or MV)
Total buffer volume per cycle
(CV or MV) 25 40 85
*CV: Column Volume Buffer cost: ~ 2 $/L
**MV: Membrane Volume
34
44. First Comments
Membrane chromatography technology leads to a much
lower volume of chromatography medium.
The buffer consumptions are considerably reduced with
membrane chromatography.
44
45. Cost simulation
for 10 and 40 batches/year during 5 years
Q membrane QyuSpeed D
Q resin
(single use) (reusable)
Loading capacity
150 2000 2000
(g protein/L-medium)
Number of use 100 1 15
Loading volume (L) 100 1500 5000 100 1500 5000 100 1500 5000
Protein loading (kg) 0,2 3 10 0,2 3 10 0,2 3 10
Total cost for
10 batches/year
during 5 years (k$)
Total cost for
40 batches/year
during 5 years (k$)
45
46. Cost simulation
for 10 and 40 batches/year during 5 years
Q membrane QyuSpeed D
Q resin
(single use) (reusable)
Loading capacity
150 2000 2000
(g protein/L-medium)
Number of use 100 1 15
Loading volume (L) 100 1500 5000 100 1500 5000 100 1500 5000
Protein loading (kg) 0,2 3 10 0,2 3 10 0,2 3 10
Total cost for
10 batches/year 731 62 299
during 5 years (k$)
Total cost for
40 batches/year 1 005 247 447
during 5 years (k$)
46
47. Cost simulation
for 10 and 40 batches/year during 5 years
Q membrane QyuSpeed D
Q resin
(single use) (reusable)
Loading capacity
150 2000 2000
(g protein/L-medium)
Number of use 100 1 15
Loading volume (L) 100 1500 5000 100 1500 5000 100 1500 5000
Protein loading (kg) 0,2 3 10 0,2 3 10 0,2 3 10
Total cost for
10 batches/year 1 025 486 477
during 5 years (k$)
Total cost for
40 batches/year 1 458 1 946 856
during 5 years (k$)
47
48. Cost simulation
for 10 and 40 batches/year during 5 years
Q membrane QyuSpeed D
Q resin
(single use) (reusable)
Loading capacity
150 2000 2000
(g protein/L-medium)
Number of use 100 1 15
Loading volume (L) 100 1500 5000 100 1500 5000 100 1500 5000
Protein loading (kg) 0,2 3 10 0,2 3 10 0,2 3 10
Total cost for
10 batches/year 1 328 1 796 670
during 5 years (k$)
Total cost for
40 batches/year 2 160 7 185 1 628
during 5 years (k$)
48
49. Cost simulation
for 10 and 40 batches/year during 5 years
Q membrane QyuSpeed D
Q resin
(single use) (reusable)
Loading capacity
150 2000 2000
(g protein/L-medium)
Number of use 100 1 15
Loading volume (L) 100 1500 5000 100 1500 5000 100 1500 5000
Protein loading (kg) 0,2 3 10 0,2 3 10 0,2 3 10
Total cost for
10 batches/year 731 1 025 1 328 62 486 1 796 299 477 670
during 5 years (k$)
Total cost for
40 batches/year 1 005 1 458 2 160 247 1 946 7 185 447 856 1 628
during 5 years (k$)
49
50. Other Comments
Membrane chromatography technology leads to a much
lower volume of chromatography medium.
The buffer consumptions are considerably reduced with
membrane chromatography.
Single use membrane chromatography is attractive with
small to medium loading volumes, when small number of
batches/year.
QyuSpeed D is attractive with medium to large loading
volumes, whatever small or high number of batches/year.
QyuSpeed D is highly cost effective thanks to its
regeneration capability.
50
51. Conclusion
QyuSpeed D can replace current AEX
chromatography steps in your DSP.
Very efficient removal of DNA, Viruses,
HCP, even at high conductivities.
QyuSpeed D is a highly cost effective
technology thanks to its regeneration
capability.
51
You can see the cut of one hollow fiber. The flow direction is from inside to outside. The flow will go through the micropore structure of the membrane . Inside each micropore you have grafted chains of poly glycidyl methacrylate. And on each chain, several ligands DEA are attached. The ligand density is very high: around 0.6 mmol/mL-ads. \n
The separation mechanism is very simple. The protein solution goes inside the hollow fiber and will permeate through the membrane structure from inside to oustide. The grafted chains with the DEA ligands offer multipoint adsorption leading to a high dynamic binding capacity. \n
QyuSpeed D can be used in flow through mode to bind impurities, negatively charged, from your protein or Mab solutions: DNA, Viruses, HCP, Prion, Endotoxin.\nPlease note that it is also possible to use our QyuSpeed D membrane in bind-elute mode to bind and concentrate specific proteins such as FVIII, fibrinogen, FIX, etc. \n
Already 3 sizes are available. 0.6 mL MV able to process up to 8 mL/min, etc&#x2026; Our 5L module will be available end of 2012. The length of the 550 mL module is only 315 mm, proving the compact design of QyuSpeed D modules.\n
\n
\n
\n
The main difference is regarding the mass transfer: convection for membrane and diffusion for resin\nDBC is independant of the flow rate unlike resine which needs a high enough contact time.\nFlow rates are much higher with membrane 5-20 MV*/min vs. 0.5-2 CV**/min. \nVery high flow rates w/o high pressure drop.\n
\n
\n
\n
Here you can see a big table summarising the performances and characteristics of the main AEX membranes on the market an also for a standard Q resin. \n
Even if DBC for BSA is used as a standard specification given by all suppliers, in the &#xAB;&#xA0;real life&#xA0;&#xBB; we are more interested in DNA, HCP and virus removal capacities.\nAt low conductivity, standard AEX resin and membrane 1 show poor DNA DBC whereas Membrane 2, Membrane 3 and QyuSpeed D show similar performances. \nIf the conductivity is higher, around 15 mS/cm, Q resin and Membrane 2 do not work properly. Membrane 1 must be replaced by another type of membrane from the same supplier. Membrane 3 and QyuSpeed D can maintain very high performances.\n
All technologies have similar performances which proves the efficiency of AEX chromatography to remove viruses. \n
\n
This is the main difference between QyuSpeedD and the other membranes on the market. QyuSpeedD can be regenerated simply like a resin. Asahi already validated 10 times regeneration with no change in BSA DBC. Of course, like for the resins, it is up to the customer to validate more regeneration cycles if necessary. Internally we could go up to 100 regenerations!\n
\n
\n
This slide can simply summarise the previous list with all advantages. One large column can be replaced by one or a few compact QyuSpeedD modules.\n
The objective of this cost simulation is to determine in which situations, Q resin, Single use AEX membrane or QyuSpeed D are the most attractive.\n
\n
\n
First, we estimated the buffer volumes needed for each technologies. We used data given by the suppliers.\nThe process steps are as follow: &#x2026;&#x2026;\n
So we need 25 CV for the Q resin, 40 MV for the single use Q membrane and 85 MV for QyuSpeed D. Of course, the buffer volumes can be optimized during a validation study.\n
\n
Then, we calculated all the costs for 1 cycle. \nWe used the following loading capacities: 150 g/L for the Q resin and 2000 g/L for the Q membrane and QyuSpeed D. \nWe considered 3 different batch volumes 100, 1500, 5000 L and therefore 3 different protein loadings according to the Mab concentration of 2 g/L: 0,2, 3 and 10 kg.\n
Then, we calculated the resin and membrane volumes needed. As already said previously, Membrane chromatography technology leads to a much lower volume of chromatography medium.\n
According to the resin and membrane volumes we calculated the costs of each technologies. \n
According to the resin and membrane volume we also calculated the buffer volumes needed. The buffer consumptions are considerably reduced with membrane chromatography.\n
According to the buffer volumes we calculated the buffer costs which are logicaly considerably reduced too.\n
We aslo estimated the labor times for each technology: 5h for the Q resin, 1,5h for the single use Q membrane and 2,5h for Qyuspeed D.\n
According to the labor times we calculated the labor costs.\n
Finaly we estimated the hardware costs and validation costs. These estimations are subjective but nevetheless we believe they are representive of the costs.\nNote that the validation cost for QyuSpeed D is half of the validation cost for Q resin because only the regeneration step must be validated. \n
So, we could find all the costs associated to 1 cycle.\n
\n
We did the cost simulation for for 10 and 40 batches/year during 5 years. We considered 100 uses for the Q resin and 15 uses for the QyuSpeedD.\n
For 100L batch volume or 0,2 kg protein loading, the single use Q membrane is the most cost effective, 2nd position QyuSpeed D and last position the Q resin.\n
For 1500L batch volume or 3 kg protein loading, there are 2 main results:\nwith 10 batches/year, Q membrane single use and QyuSpeed D are both the most cost effective. \nwith 40 batches/year, QyuSpeed D is the most cost effective, 2nd position the Q resin and last position the single use Q membrane.\n
For the largest volume, 5000L batch volume or 10 kg protein loading, QyuSpeed D is the most cost effective, 2nd position the Q resin and last position the single use Q membrane.\n