Polishing of monoclonal antibodies using a polymer grafted ...€¦ · Polishing of monoclonal...
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Polishing of monoclonal antibodies using a polymer grafted cation exchanger Anna Åkerblom, Jesper Hansson, Bengt Westerlund, Per-Mikael Åberg, and Anna Heijbel GE Healthcare Bio-Sciences, Björkgatan 30, SE-75184 Uppsala, Sweden GE and GE monogram are trademarks of General Electric Company. ÄKTA, Capto, MabSelect SuRe, PreDictor, Superdex, and Tricorn are trademarks of General Electric Company or one of its subsidiaries. Eshmuno and Fractogel are trademarks of Merck KGaA. Nuvia is a trademark of Bio-Rad Laboratories Inc. Poros is a trademark of Life Technologies Corp. All other third party trademarks are the property of their respective owners. © 2014 General Electric Company – All rights reserved. First published Mar. 2014 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-75184 Uppsala, Sweden 09/2014 29-1030-53 AD Introduction A typical monoclonal antibody (MAb) purification process begins with capture on a protein A chromatography medium (resin), followed by polishing steps by for example ion exchange chromatography. The polishing steps need to overcome several challenges. Removal of aggregates, one of the major goals of the purification process, is such an obstacle. Today, higher titers (> 3 g/L) in upstream production are common. This might lead to higher aggregate levels and consequently increase the demands on downstream processing. A newly developed grafting technology, resulting in a high-capacity and high-resolution cation exchange (CIEX) medium designed for polishing of monoclonal antibodies, Capto™ S ImpAct (Fig 1), can help solve this problem. This study shows that a prototype of the medium can handle variations in upstream production, with maintained yield and aggregate removal levels. Screening of binding conditions To find the optimal binding conditions for the used MAb, static binding capacity (SBC) was measured in PreDictor™ plates, where pH and salt concentration were varied. Optimal binding, at the conditions tested, was found over a wide pH-range (pH 4.75 to 5.5), at a slightly increased ionic strength (Fig 2). The trend was confirmed in dynamic binding capacity (DBC) measurements in a packed column format (data not shown). Comparison with CIEX media on the market SBC was measured for a selected MAb (MAb A in Fig 6) using Capto S ImpAct, as well as several commercially available CIEX media, in a wide range of pH and salt concentrations. Based on SBC data, DBC was measured in triplicate runs at optimal conditions for each medium*. Figure 6 shows that Capto S ImpAct gives high DBC for a variety of different antibodies, and has better performance than the other cation exchangers tested. Optimization of elution conditions A design of experiments (DoE) approach was used to find the optimal elution strategy for the used MAb, with different aggregate content at different loads. Factors in the DoE were load, start aggregate content, gradient length, residence time during elution, and pH of the elution buffer. Binding conditions were identical for all experiments. As can be seen in the coefficient plot (Fig 4), the most significant factors for removal of aggregates were gradient length and start aggregate content. Screening of selectivity conditions A number of conditions were chosen, from the SBC data, for further evaluation of aggregate removal. It was found that pH had a significant effect on the resolution between monomers and aggregates. The best separation observed was found at pH 5. Figure 3 shows an example of a chromatogram, where 80 mg MAb/mL medium was loaded. At 90% monomer yield the aggregate level was 0.7%. The aggregate content from start was 4.2%. Conclusions Capto S ImpAct medium shows: • High resolution between aggregates and monomer at high load with high yield • High binding capacity for all tested antibodies • Higher binding capacities than five, out of five tested, commercially available cation exchangers = Pyrrolidone = Sulfonate Matrix: High-flow agarose Functional group: SO 3 - Total ionic capacity: 37 to 63 μmol (H + )/ml medium Average particle size: 50 μm Fig 1. Schematic picture of the polymer grafted Capto S ImpAct. Grafting occurs randomly to form the polymer surface extender. Fig 2. SBC measured in 50 mM sodium acetate with protein A-purified MAb. Fig 6. Comparison of dynamic binding capacity using different CIEX media. Column: Tricorn 5/50, 1 mL CV Sample: Two Mabs purified on MabSelect SuRe or commercially available polyclonal IgG (pIgG) Sample load: Until 10% breakthrough Start buffer: MAb A, pH 5.0: 50 mM sodium acetate, pH 5.0 + 50 mM NaCl (optimum for Capto S ImpAct, Eshmuno™ CPX, Fractogel™ EMD SO 3 - (M), and Poros™ XS) Start buffer: MAb A, pH 6.0: 50 mM sodium acetate, pH 6.0 (optimum for Capto SP ImpRes and Nuvia™ HR-S) Start buffer: MAb B and pIgG: 50 mM sodium acetate, pH 5.5 Flow rate: 0.19 mL/min, 5.4 min residence time System: ÄKTA system * The comparison was performed January and February, 2014, in Uppsala, Sweden. Medium: Capto S ImpAct Column: Tricorn™ 5/50, 1 mL column volume (CV) Sample: MAb purified on MabSelect SuRe™ Sample load: 80 mg MAb/mL medium Start buffer: 50 mM sodium acetate, pH 5.0 + 50 mM NaCl Elution buffer: 50 mM sodium acetate, pH 5.0 + 500 mM NaCl Flow at loading: 0.25 mL/min, 4 min residence time Flow at elution: 0.125 mL/min, 8 min residence time Gradient: Linear, 20 CV System: ÄKTA™ pure 25 Cleaning-in-place (CIP): 1 M NaOH Fig 3. Typical chromatogram. Aggregate content was analyzed with size- exclusion chromatography using Superdex™ 200 Increase 10/300 GL column. Fig 4. Coefficient plot for the aggregate content at 90% monomer yield. Positive bars have an increasing effect on the measured response when the parameter setting goes from its low to high value and vice versa for the negative bars. Fig 5. 4D contour plot showing the prediction of aggregate content at 90% monomer yield in the elution pool after gradient elution in 50 mM sodium acetate, pH 5.0 + 500 mM NaCl. pH Concentration (mM) 350 300 250 200 150 100 50 0 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 0–350 mM NaCl pH 4.5–6.0 > 100 mg/mL 100–70 mg/mL 70–40 mg/mL mAU Aggregate (%) ml 30 25 20 40 35 15 10 5 0 3000 2750 2500 1750 1500 1250 1000 750 500 250 0 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 Conductivity (mS/cm) 175 150 125 100 75 50 25 0 % aggregate in fraction CIP UV 280 nm Conductivity Pool with 90% monomer yield and 0.7% aggregate content Gradient (CV) Aggregate (%) 20 18 16 14 12 10 Gradient (CV) 18 16 14 12 10 Gradient (CV) Load (mg/mL) = 80 Load (mg/mL) = 65 Load (mg/mL) = 50 18 16 14 12 10 Aggregate (%) Aggregate (%) Residence time (min) = 4 Residence time (min) = 6 Residence time (min) = 8 6.5 6.0 5.5 5.0 4.5 6.5 6.0 5.5 5.0 4.5 6.5 6.0 5.5 5.0 4.5 1.6 1.4 1.4 1.2 1 1 1 1.6 1.4 1.2 1.2 1 1 1 1 1 0.8 0.8 1.6 1.4 1.4 1.4 1.2 1.2 1.2 1.2 1.2 1.2 1 0.8 0.8 0.8 0.8 0.8 Aggregate content at 90% monomer recovery (%) Load Start aggregate content Gradient lenght Residence time pH elution buffer Load*Load Start aggregate content*Gradient lenght Start aggregate content*Residence time Gradient lenght*Residence time 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 N = 29 DF = 19 R 2 = 0.976 Q 2 = 0.947 RSD = 0.05524 Conf. level = 0.95 DBC 10% breakthrough (mg/mL) 120 100 80 60 40 20 0 Capto S ImpAct Capto SP ImpRes Eshmuno CPX Fractogel EMD SO 3 - (M) Poros XS Nuvia HR-S MAb A, pH 5.0 MAb A, pH 6.0 MAb B, pH 5.5 pIgG, pH 5.5 Even at high load (80 mg MAb/mL) and high aggregate content, 7%, the DoE model predicts that the aggregate level is reduced to < 1% at a monomer yield over 90% (Fig 5). This was verified with extra experiments. Host cell protein levels were low in the start sample and no significant model for removal could be found.
Transcript of Polishing of monoclonal antibodies using a polymer grafted ...€¦ · Polishing of monoclonal...
Polishing of monoclonal antibodies using a polymer grafted cation exchanger Anna Åkerblom, Jesper Hansson, Bengt Westerlund, Per-Mikael Åberg, and Anna Heijbel GE Healthcare Bio-Sciences, Björkgatan 30, SE-75184 Uppsala, Sweden
GE and GE monogram are trademarks of General Electric Company. ÄKTA, Capto, MabSelect SuRe, PreDictor, Superdex, and Tricorn are trademarks of General Electric Company or one of its subsidiaries.Eshmuno and Fractogel are trademarks of Merck KGaA. Nuvia is a trademark of Bio-Rad Laboratories Inc. Poros is a trademark of Life Technologies Corp. All other third party trademarks are the property of their respective owners.© 2014 General Electric Company – All rights reserved. First published Mar. 2014All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-75184 Uppsala, Sweden 09/2014 29-1030-53 AD
IntroductionA typical monoclonal antibody (MAb) purification process begins with capture on a protein A chromatography medium (resin), followed by polishing steps by for example ion exchange chromatography. The polishing steps need to overcome several challenges. Removal of aggregates, one of the major goals of the purification process, is such an obstacle. Today, higher titers (> 3 g/L) in upstream production are common. This might lead to higher aggregate levels and consequently increase the demands on downstream processing.
A newly developed grafting technology, resulting in a high-capacity and high-resolution cation exchange (CIEX) medium designed for polishing of monoclonal antibodies, Capto™ S ImpAct (Fig 1), can help solve this problem. This study shows that a prototype of the medium can handle variations in upstream production, with maintained yield and aggregate removal levels.
Screening of binding conditionsTo find the optimal binding conditions for the used MAb, static binding capacity (SBC) was measured in PreDictor™ plates, where pH and salt concentration were varied. Optimal binding, at the conditions tested, was found over a wide pH-range (pH 4.75 to 5.5), at a slightly increased ionic strength (Fig 2). The trend was confirmed in dynamic binding capacity (DBC) measurements in a packed column format (data not shown).
Comparison with CIEX media on the marketSBC was measured for a selected MAb (MAb A in Fig 6) using Capto S ImpAct, as well as several commercially available CIEX media, in a wide range of pH and salt concentrations. Based on SBC data, DBC was measured in triplicate runs at optimal conditions for each medium*.
Figure 6 shows that Capto S ImpAct gives high DBC for a variety of different antibodies, and has better performance than the other cation exchangers tested.
Optimization of elution conditionsA design of experiments (DoE) approach was used to find the optimal elution strategy for the used MAb, with different aggregate content at different loads.
Factors in the DoE were load, start aggregate content, gradient length, residence time during elution, and pH of the elution buffer. Binding conditions were identical for all experiments. As can be seen in the coefficient plot (Fig 4), the most significant factors for removal of aggregates were gradient length and start aggregate content.
Screening of selectivity conditionsA number of conditions were chosen, from the SBC data, for further evaluation of aggregate removal. It was found that pH had a significant effect on the resolution between monomers and aggregates. The best separation observed was found at pH 5. Figure 3 shows an example of a chromatogram, where 80 mg MAb/mL medium was loaded. At 90% monomer yield the aggregate level was 0.7%. The aggregate content from start was 4.2%.
ConclusionsCapto S ImpAct medium shows:
• High resolution between aggregates and monomer at high load with high yield
• High binding capacity for all tested antibodies
• Higher binding capacities than five, out of five tested, commercially available cation exchangers
= Pyrrolidone = Sulfonate
Matrix: High-flow agarose
Functional group: SO3-
Total ionic capacity: 37 to 63 μmol (H+)/ml medium
Average particle size: 50 μm
Fig 1. Schematic picture of the polymer grafted Capto S ImpAct. Grafting occurs randomly to form the polymer surface extender.
Fig 2. SBC measured in 50 mM sodium acetate with protein A-purified MAb.
Fig 6. Comparison of dynamic binding capacity using different CIEX media.
Column: Tricorn 5/50, 1 mL CV
Sample: Two Mabs purified on MabSelect SuRe or commercially available polyclonal IgG (pIgG)
Sample load: Until 10% breakthrough
Start buffer: MAb A, pH 5.0: 50 mM sodium acetate, pH 5.0 + 50 mM NaCl (optimum for Capto S ImpAct, Eshmuno™ CPX, Fractogel™ EMD SO3
- (M), and Poros™ XS)
Start buffer: MAb A, pH 6.0: 50 mM sodium acetate, pH 6.0 (optimum for Capto SP ImpRes and Nuvia™ HR-S)
Start buffer: MAb B and pIgG: 50 mM sodium acetate, pH 5.5
Flow rate: 0.19 mL/min, 5.4 min residence time
System: ÄKTA system
* The comparison was performed January and February, 2014, in Uppsala, Sweden.
Medium: Capto S ImpAct Column: Tricorn™ 5/50, 1 mL column volume (CV)Sample: MAb purified on MabSelect SuRe™Sample load: 80 mg MAb/mL mediumStart buffer: 50 mM sodium acetate, pH 5.0 + 50 mM NaClElution buffer: 50 mM sodium acetate, pH 5.0 + 500 mM NaClFlow at loading: 0.25 mL/min, 4 min residence timeFlow at elution: 0.125 mL/min, 8 min residence timeGradient: Linear, 20 CVSystem: ÄKTA™ pure 25Cleaning-in-place (CIP): 1 M NaOH
Fig 3. Typical chromatogram. Aggregate content was analyzed with size-exclusion chromatography using Superdex™ 200 Increase 10/300 GL column.
Fig 4. Coefficient plot for the aggregate content at 90% monomer yield. Positive bars have an increasing effect on the measured response when the parameter setting goes from its low to high value and vice versa for the negative bars.
Fig 5. 4D contour plot showing the prediction of aggregate content at 90% monomer yield in the elution pool after gradient elution in 50 mM sodium acetate, pH 5.0 + 500 mM NaCl.
pH
Concentration (mM)350300250200150100500
6.0
5.9
5.8
5.7
5.6
5.5
5.4
5.3
5.2
5.1
5.0
4.9
4.8
4.7
4.6
4.5
0–350 mM NaCl
pH 4
.5–6
.0
> 100 mg/mL
100–70 mg/mL
70–40 mg/mL
mAU
Aggr
egat
e (%
)
ml302520 4035151050
3000
2750
2500
1750
1500
1250
1000
750
500
250
0
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Con
duct
ivity
(mS/
cm)
175
150
125
100
75
50
25
0
% aggregate in fraction
CIP
UV 280 nm Conductivity Pool with 90% monomer yieldand 0.7% aggregate content
Gra
dien
t (C
V)
Aggregate (%)
20
18
16
14
12
10
Gra
dien
t (C
V)
18
16
14
12
10
Gra
dien
t (C
V)
Load
(mg/
mL)
= 8
0Lo
ad (m
g/m
L) =
65
Load
(mg/
mL)
= 5
0 18
16
14
12
10
Aggregate (%) Aggregate (%)
Residence time (min) = 4 Residence time (min) = 6 Residence time (min) = 8
6.56.05.55.04.56.56.05.55.04.5 6.56.05.55.04.5
1.6
1.4
1.4
1.2
1
1
1
1.6
1.4
1.2
1.2
1
1
11
1
0.8
0.8
1.6
1.4
1.4
1.4
1.2
1.2
1.2
1.2
1.2
1.2
1
0.80.8
0.8
0.8
0.8
Aggr
egat
e co
nten
t at 9
0% m
onom
er re
cove
ry (%
)
Load
Start
aggregate
conte
nt
Gradient le
nght
Residence
time
pH elution buffe
r
Load*L
oad
Start
aggregate
conte
nt*Gra
dient lenght
Start
aggregate
conte
nt*Resid
ence tim
e
Gradient le
nght*Resid
ence tim
e
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
N = 29DF = 19R2 = 0.976Q2 = 0.947RSD = 0.05524Conf. level = 0.95
DBC
10%
bre
akth
roug
h (m
g/m
L)
120
100
80
60
40
20
0Capto SImpAct
Capto SPImpRes
Eshmuno CPX Fractogel EMD SO3
- (M) Poros XS Nuvia HR-S
MAb A, pH 5.0 MAb A, pH 6.0 MAb B, pH 5.5 pIgG, pH 5.5
Even at high load (80 mg MAb/mL) and high aggregate content, 7%, the DoE model predicts that the aggregate level is reduced to < 1% at a monomer yield over 90% (Fig 5). This was verified with extra experiments. Host cell protein levels were low in the start sample and no significant model for removal could be found.
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