a comparison of protein a chromatographic stationary phases
TRANSCRIPT
Zhuo Liu Process Development
KBI Biopharma Inc.
2 Triangle Dr. RTP, NC 27709
BioProcess International Conference, Boston, MA, USA. October 26-29, 2015
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• Objective and scope of work
• Properties of Protein A resins and model mAbs
• Performance characteristics comparison
• Conclusions
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• Objective: compare leading Protein A resins in terms of their performance characteristics
• Five different Protein A resins were compared using an effective methodology developed from previous work
• Resin performance characteristics comparison with four different mAbs • Static binding capacity
• Dynamic binding capacity
• Productivity
• Elution pH
• Purification process performance including HCP and elution column volume
Liu,Z., Mostafa, S.S., Shukla, A.A., Biotechnol. and Appl. Biochem., 62 (1), 37-47, 2015.
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Resin Vendor Matrix Ligand Modified Protein A
domain Mean particle
size (µm)
MabSelect SuReTM GE Healthcare Highly cross-linked
agarose
Alkali-stabilized
rProtein A B domain 85
MabSelect SuReTM LX GE Healthcare Highly cross-linked
agarose
Alkali-stabilized
rProtein A B domain 85
ToyopearlTM AF-rProtein A HC-650F† Tosoh Polymethacrylate
Alkali-stabilized
rProtein A C domain 45
EshmunoTM A† EMD Millipore Cross-linked
Polyvinyl Ether
Alkali-stabilized
rProtein A C domain 50
AmsphereTM A3† JSR Life Sciences
Polymethacrylate Alkali-stabilized
rProtein A C domain 50
† Not included in previous paper TM Registered trademark of each vendor
mAb IgG Class Titer (g/L)
mAb1 IgG1 1.08
mAb2 IgG1 2.58
mAb3 IgG4 3.90
mAb4 IgG1 10.0
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Static binding capacity is mAb dependent for each resin.
MabSelect SuRe and Eshmuno A have comparable static binding capacities.
MabSelect SuRe LX, HC-650F and Amsphere A3 have comparable static binding capacities, which are 10-40 % higher than MabSelect SuRe and Eshmuno A. mAb1 mAb2 mAb3 mAb4
50
60
70
80
90
100
110
120
130
SB
C (
g/L
)
MabSelec SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
Batch mode with Protein A purified material at concentration ~ 10 g/L Binding buffer: 20mM Tris, 150mM NaCl, pH 7.5 Static binding capacity determined by Langmuir isotherm linear fit
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1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
DB
C (
g/L
)
Residence Time (min)
MabSelect SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
mAb1
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80mAb2
DB
C (
g/L
)
Residence Time (min)
MabSelect SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80mAb4
DB
C (
g/L
)
Residence Time (min)
MabSelect SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80mAb3
DB
C (
g/L
)
Residence Time (min)
MabSelect SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
DBC is mAb dependent for each resin.
Amsphere A3 resin showed the highest binding capacities for all flow rates with three tested mAbs.
High static binding capacity for MabSelect SuRe LX and HC-650F did not translate into a high dynamic binding capacity.
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• Important consideration during commercial manufacturing focusing on minimizing suite time for DSP
• Increased emphasis on higher productivity in DSP with high titer achieved in cell culture
Productivity =
( Amount of protein purified
Unit of resin
Unit time
)
Rpv = 1
Lc ( 1
C0 • Ul +
N
Qd • Ue )
Rpv: volumetric production rate (g/L/h) Lc: bed height (cm) C0: feed load titer (g/L) Ul: linear flow rate for the load (cm/h) N: number of CVs for non-load steps (unitless) Qd: dynamic binding capacity (g/L) Ue: linear flow rate for non-load steps (cm/h) Qd
∞: DBC at long residence times (g/L) τd: residence time constant (h) τ: residence time (h)
Qd =
Qd∞ • τ
τd + τ
No pressure constrains assumed for all resins with maximum operational flow rate used for non-load steps
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Productivity surface showed a optimum respect to linear velocity and column length.
Amsphere A3 demonstrated the highest optimum productivity of 37 g/L-resin/h (~ 20% higher) among tested Protein A resins because of highest dynamic binding capacity at shorter residence time.
Other resins exhibited different optimum productivities around 23 to 30 g/L-resin/h.
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Productivity surface showed a optimum respect to linear velocity and column length.
Amsphere A3 demonstrated the highest optimum productivity of 52 g/L-resin/h (~ 25% higher) among tested Protein A resins because of highest dynamic binding capacity at all flow rates.
Other resins exhibited comparable optimum productivities around 35 to 42 g/L-resin/h.
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In general, higher productivity achieved for each resin with increase of load protein titer.
Amsphere A3 demonstrated the highest optimum productivity of 64 g/L-resin/h (~ 40% higher) among tested Protein A resins.
Other resins exhibited comparable optimum productivities around 41 to 46 g/L-resin/h.
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In general, higher productivity achieved for each resin with increase of load protein titer.
Amsphere A3 demonstrated the highest optimum productivity of 93 g/L-resin/h (~ 50% higher) among tested Protein A resins.
Other resins showed comparable optimum productivities around 60 g/L-resin/h.
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3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
Elu
tio
n p
H
mAb1
mAb2
mAb3
mAb4
Step Gradient Elution
Similar elution pHs were observed for any given resin with various mAbs.
MabSelect SuRe, SuRe LX, Eshmuno A and Amsphere A3 all had comparable milder elution pH.
HC-650F demonstrated the lowest elution pH among different resins.
Milder elution pH is better for pH sensitive mAbs.
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Binding buffer: 20mM Tris, 150mM NaCl, pH7.5 with 30g/L loading
Pre-elution wash: 50mM Acetate pH 5.0 to 5.8
Elution pH determined from step gradient elution study
Comparable yield and purity (SEC) achieved for different resins with same mAb
HCP determined by HCP ELISA
MabSelect SuRe, SuRe LX, Eshmuno A and Amsphere A3 performed comparably well in terms of HCP clearance.
HC-650 F resulted in the lowest HCP reduction compared to other resins for the mAbs tested.
mAb1 mAb2 mAb3 mAb40
1000
2000
3000
8000
9000
10000
HC
P L
evel (p
pm
)
MabSelec SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
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Binding buffer: 20mM Tris, 150mM NaCl, pH 7.5 with 30 g/L loading Pre-elution wash: 50mM Acetate pH 5.0 to 5.8 Elution pH determined from step gradient elution study
mAb1 mAb2 mAb3 mAb40
1
2
3
4
5
Elu
tio
n C
V
MabSelec SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
The number of elution column volumes (CVs) required for the product elution was fairly similar for four Protein A resins with variation between 1.5 and 2.5 CVs.
Eshmuno A consistently demonstrated higher elution CVs compared to other resins for the mAbs tested.
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Resin Static
binding capacity
Dynamic binding capacity
Productivity Elution pH HCP
clearance
Elution column volume
MabSelect SuRe ++ ++ ++ ++ +++ +++
MabSelect SuRe LX +++ ++ ++ +++ +++ +++
Toyopearl AF-rProtein A HC-650F +++ ++ ++ + ++ ++
Eshmuno A ++ ++ ++ +++ +++ +
Amsphere A3 +++ +++ +++ +++ +++ ++
• This work comprehensively compared five different Protein A resins in terms of performance characteristics with four different model mAbs.
• Amsphere A3 resin exceeds in most aspects in this comparison study, which makes it a good Protein A resin candidate for platform monoclonal antibody purification in downstream process development and manufacturing applications.
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Process Development • Sigma S. Mostafa Ph.D.
• Carnley Norman Ph.D.
• Cartney Barringer
Analytical Development • Jimmy Smedley III Ph.D.
• Helena Gaweska Ph.D.
• Adam Barnes
• Michael Pollock
• Tracy Cheung Ph.D.
• Abhinav A. Shukla Ph.D.
Senior VP, Process Development and Manufacturing
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