intelligent design of the ion-exchange analytical column
TRANSCRIPT
Intelligent Design of the Ion-Exchange Analytical Column for Monoclonal Antibody CharacterizationIntelligent Design of the Ion-Exchange Analytical Column for Monoclonal Antibody CharacterizationYuanxue Hou, Srinivasa Rao, Yury Agroskin, and Chris PohlDionex Corporation, Sunnyvale, CA, USAYuanxue Hou, Srinivasa Rao, Yury Agroskin, and Chris PohlDionex Corporation, Sunnyvale, CA, USA
IntroductIonThe dramatic growth of the monoclonal antibody (MAb) therapeutics market has generated enormous demand for more powerful analytical tools for protein characterization and fractionation. A broad range of analytical tools has evolved to meet the requirements of the biotechnology industry. Among them, ion-exchange chromatography (IEC) is one of the most widely used techniques for the characterization and analysis of MAb charge variants. For example, the ProPac® WCX-10 analytical column is well known for high-resolution, high-peak capacity MAb separations.
In this presentation, the authors discuss the latest development of a new MAb column–the MAbPac SCX-10–designed as a complimentary addition to existent ion-exchange products providing high resolution and orthogonal selectivity for various proteins and MAb charge variant characterization.
The effects of separation media formulations and process parameters were also investigated. The main objective was to develop a new selectivity and enhanced peak capacity by optimizing the column’s chemical design from raw resin synthesis to the final functionalized step. Various polymerization techniques, including the atom transfer rapid polymerization (ATRP) approach, were applied in an attempt to build a well-controlled and uniform ion-exchange separation surface on a highly, cross-linked, polymeric resin. The separation of both protein mixtures and real MAb samples are discussed to emphasize the utility of this MAb column.
The reproducibility and robustness of the MAbPac SCX-10 column is demonstrated. This poster also discusses the effect of process optimization and the polymerization approach on MAb separation performance.
ExpErImEntalInstrumentDionex ICS-3000 ion chromatography system consisting of: DP Dual gradient pump ASI-100 Autosampler AD25 Absorbance detector STH 585 Temperature-controlled column compartment Chromeleon® Chromatography Data System software from Dionex Corporation.
2 OptimizationoftheSeparationPowerof1-DNanoLCAnalysisofProteomicsSamples
IntelligentDesignoftheIon-ExchangeAnalyticalColumnforMonoclonalAntibodyCharacterization
rEsultsDesign of Separation MediaA novel, nonporous, highly cross-linked styrene-type polymeric media was developed. A one-step surface modification process creates strong hydrophilic properties of spherical DVB particulates. These particles exhibit very low nonspecific binding during protein separation.
The second critical part of the design is a strong cation-exchange layer. There are many ways to introduce functional groups. In this work, ATRP principles, grafting-from, were applied to better control polymeric chain length and density of functional groups.
Figure 1. MAbPac SCX-10 separation media design: example separations of MAb 1, 2, 3, and 4 are shown.
Hydrophilic Boundary Layer Modification Three strategies were evaluated: oxidation, encapsulation, and in situ modification. Each approach produced different degrees of hydrophilicity, resulting in different column performance in terms of peak width at half height (PWHH), column capacity, and number of peaks for protein mix separation. As can be seen in Figure 2, in situ modification provides sharper peaks and higher peak capacity compared to the other approaches.
Figure 2. Comparison of the applied approaches for the creation of a hydrophilic layer.
0 20.0 40.0 58.7–0.0003
0.0115
AU
Minutes6 12 18
0
10
15 20 30 3525 40–5.0
50
Minutes
MAb 1 MAb 2
MAb 3 MAb 4
0 20 40 67Minutes
–5
35
mAU mAU
mAU
Minutes
HydrophilicBoundary Layer Grafted Linear
Ion-Exchange Layer
27501
Highly Cross-linked
Polymeric Bead
MMinutes
27502
–0.0015
0.0142
AU
–0.0015
0.0154
AU
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0–0.0012
0.0132
AU
Minutes
In situ modification
Oxidation
Encapsulation
2
1
3
2
1
3
2
1
3
PWHH: 0.17
PWHH: 0.11
PWHH: 0.10
Column: 4 × 250 mmA: 10 mM Na2HPO4, pH = 6.0B: 10 mM Na2HPO4, 1.0 M NaCl, pH = 6.0
t (min) %A %B 0.0 100 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nmSample: 1. Ribonuclease A (Rib) 2. Cytochrome C (Cyt C) 3. Lysozyme (Lys)
3
ATRP Grafting Process: Creation of the Functional Layer (IEX)The first series of experiments were directed to develop a new orthogonal selectivity compared to current ProPac columns. This was achieved by switching from a weak cation mode (WCX) to a strong cation exchanger, like sulfonic groups (SO3
–), and by adjusting monomer composition.
Figure 3. Selectivity of MAbPac SCX column when compared to the ProPac WCX column. The order of elution of the analytes for the MAbPac SCX column is ribonuclease A followed by cytochrome C. This is reversed for the ProPac WCX column.
In another example, both MAbPac SCX-10 and ProPac SCX-10 columns have the same functionality, but the type and the structure of the resin surface is differently designed, resulting in different column performance as shown in Figure 4.
Figure 4. Comparison of MAbPac SCX-10 vs ProPac SCX-10 columns: separation of protein mixture.
27503
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5
–0.0015
0.0127
AU
–0.0020
0.0120
AU
Minutes
ProPac WCX-10
MAbPac SCX-10
3
2
1
3
2
1
Column: 4 × 250 mmA: 20 mM MES, pH = 6.4B: 20 mM MES, 1.0 M NaCl, pH = 6.4
t (min) %A %B 0.0 100 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nmSample: 1. Ribonuclease A (Rib) 2. Cytochrome C (Cyt C) 3. Lysozyme (Lys)
27504
–0.0010
0.0121
AU
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.5–0.0013
0.0143
AU
Minutes
ProPac SCX-10
MAbPac SCX-10
3
2
1
3
2
1
Column: 4 × 250 mmA: 20 mM MES, pH = 6.4B: 20 mM MES, 1.0 M NaCl, pH = 6.4
t (min) %A %B 0.0 00 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nmSample: 1. Ribonuclease A (Rib) 2. Cytochrome C (Cyt C) 3. Lysozyme (Lys)
4 OptimizationoftheSeparationPowerof1-DNanoLCAnalysisofProteomicsSamples
IntelligentDesignoftheIon-ExchangeAnalyticalColumnforMonoclonalAntibodyCharacterization
The advantages of the ATRP method are well known. Here, an attempt was made to implement the grafting process with a controlled degree of polymerization and polymeric functional groups density.
The graft density can be regulated by the ratio of surface initiator to resin amount (Rd). As shown in Figure 5, various grafting densities (Rd) affect the separation capacity and efficiencies of the protein. For example, higher Rd values reduce peak width of lysozyme.
Figure 5. Effect of graft density.
27505
–0.00050
0.00493
AU
–0.00077
0.00347
AU
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.9–0.00113
0.00724
AU
Minutes
Rd 3
Rd 1
Rd 2 PWHH: 0.14
PWHH: 0.18
PWHH: 0.11
2
1 3 2
1
3
2
1
3
Column: 4 × 250 mmA: 10 mM Na2HPO4, pH = 6.0B: 10 mM Na2HPO4, 1.0 M NaCl, pH = 6.0
t (min) %A %B 0.0 00 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nmSample: 1. Ribonuclease A (Rib) 2. Cytochrome C (Cyt C) 3. Lysozyme (Lys)
Rd 1 < Rd 2 < Rd 3
5
Degree of polymerization (DP) is another important parameter for protein separation. A ratio between monomer amount and ATRP initiator (DP = M/I, mol/mol) is a traditional way to describe the length of polymeric chains.
The isocratic and gradient test results showed that the column capacity can be controlled by adjusting the graft length without affecting the selectivity of the protein mix.
Figure 6. Effect of graft length.
27506
–0.0005
0.0176
AU
–0.0018
0.0251
AU
0 4.0 8.0 12.0 16.0 20.6–0.0011
0.0247
Minutes
PWHH: 0.10
PWHH: 0.09
M/I: 100
M/I: 50
PWHH: 0.09 M/I: 200
2 3
1
2 3
1
2
3
1
–0.0006
0.0137
AU
–0.0010
0.0100
AU
–0.01 1.00 2.00 3.00 4.00 5.05–0.0020
0.0120
AU
Minutes
Isocratic Test of Cyt C Gradient Test of Protein Mix
Cyt C
M/I: 100
M/I: 50
M/I: 200
Column: 4 × 250 mmA: 10 mM Na2HPO4, pH = 6.0B: 10 mM Na2HPO4, 1.0 M NaCl, pH = 6.0
t (min) %A %B 0.0 100 0 20.25 52 48
AU
Flow Rate: 1.0 mL/minλ: 254 nmSample: 1. Ribonuclease A (Rib) 2. Cytochrome C (Cyt C) 3. Lysozyme (Lys)
6 OptimizationoftheSeparationPowerof1-DNanoLCAnalysisofProteomicsSamples
IntelligentDesignoftheIon-ExchangeAnalyticalColumnforMonoclonalAntibodyCharacterization
It was established that by reducing the monomer amount and keeping the remaining parameters the same, MAb separation can be speeded up without compromising selectivity.
Figure 7. Effect of the monomer/resin ratio (M/R).
rEproducIbIlIty and ruggEdnEss pHStabilityStudyThe column was treated at ambient temperature under basic (pH~12) conditions for 7 h and then under acidic (2.5% H2SO4) conditions for 2 h. The chromatographic test was conducted before and after the treatment. The performance results of protein mix did not show any degradation in terms of the retention time and peak width (Figure 8).
Figure 8. Characterization: pH stability study.
27507
–0.0009
0.0315
AU
0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 44.0 48.0 51.1–0.0011
0.0280
AU
Minutes
M/R = 0.125
M/R = 1.0
Column: 4 × 250 mmA: 20 mM MES, 60 mM NaCl, pH = 5.5B: 20 mM MES, 240 mM NaCl, pH = 5.5
t (min) %A %B 2.0 77 23 60.0 57 43
Flow Rate: 1.0 mL/minλ: 280 nmSample: MAb (PDL)
27508
0 3 6 9 12 15 18 21–0.0010
0.0336
AU
Minutes
7.09 11.040.00055
0.00327
AU
Minutes
After treatment
Before treatment
PWHH: 0.10
PWHH: 0.10
Lys Cyt C Rib
Column treated with 10 mM NaOH (pH~12) for 7 h, then with 2.5% H2SO4 for 2 h at RTwith a flow rate of 1 mL/min
7
TemperatureStabilityStudyA column was treated at 80 °C with MES buffer (pH~5.5) for 3 h. A functional test was conducted before and after the treatment. The chromatographic performance of the protein mix did not show any significant degradation in terms of the retention time and peak width (Figure 9).
Figure 9. Characterization: temperature stability study.
ReproducibilityEvaluationColumn reproducibility (σ0) is a part of any column quality characterization• Column-to-column reproducibility component (σcol) represents: – Column packing processes with separation media – Hardware supplies• Lot-to-lot reproducibility component (σlot) represents: – Polymerization processes – Raw material supplies – Separation media properties
■ Physical/chemical properties of bed support ■ Chemical properties of particles’ surface or latex
The total column reproducibility can be calculated by using Formula 1:Formula 1: σ0
2 = σcol2 + σlot
2 + σer2
Where σer is measurement/experimental error.
Lot-to-lot reproducibility is one of the most important parameters for MAb separation. This study was set up in the most vigorous terms. Each batch was produced starting from separate raw resin synthesis through all functionalizaion steps. As the functional test shows in Figure 10, the chromatographic performance exibits excellent lot-to-lot reproducibility with RSD < 2% in term of retention time, and the peak width at half height.
Figure 10. Reproducibility showing lot-to-lot consistency.
27509
0 5.0 10.0 15.0 20.0 22.8–0.0027
0.0309
AU
Minutes
After treatment
Before treatment
PWHH: 0.09
PWHH: 0.09
Lys
Cyt CRib
6.48 8.00 10.230.00008
0.00249
AU
Minutes
Column heated at 80 °C h with a buffer containing 20 mM MES, 240 mM NaCl, pH 5.5 with a flow rate of 1 mL/min
27510
–0.00027
0.00549
AU
1 - 25.224
2 - 28.1243 - 32.147
–0.00006
0.00492
AU
1 - 24.497
2 - 27.2973 - 31.304
–0.1 4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 44.0 48.0 53.9–0.00017
0.00511
AU
Minutes
1 - 25.247
2 - 28.0743 - 32.090
I
II
III
Column: 4 × 250 mmA: 20 mM MES, 60 mM NaCl, pH = 5.5B: 20 mM MES, 300 mM NaCl, pH = 5.5
t (min) %A %B 2.0 85 15 52.0 63.56 36.44
Flow Rate: 1.0 mL/minλ: 280 nmSample: MAb (PDL)
8 OptimizationoftheSeparationPowerof1-DNanoLCAnalysisofProteomicsSamples
IntelligentDesignoftheIon-ExchangeAnalyticalColumnforMonoclonalAntibodyCharacterization
Column-to-ColumnReproducibilityAs shown in Figure 11, the excellent column-to-column reproducibility verified the column packing methods (RSD: 0.52).
Figure 11. Reproducibility showing column-to-column consistency.
Run-to-RunReproducibilityWhen 100 runs of Cyt C test were performed continuously on the same column, excellent reproducibility (RSD: 0.74) was obtained.
Figure 12. Reproducibility showing run-to-run consistency.
EffectsofChromatographicTestConditionsMobile phase can be effectively used in method optimization. Different buffer solutions result in variations of protein resolution, such as ribonuclease (Rib), Cytochrome C (Cyt C), even with the same pH and salt gradient.
Figure 13. Effect of mobile phase.
27511
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56–0.0043
0.0084
AU
Minutes
IV
IIIII I
Column: 4 × 250 mmA: 20 mM MES, 60mM NaCl, pH = 5.5B: 20 mM MES, 300 mM NaCl, pH = 5.5 t (min) %A %B 2.0 85 15 52.0 63.56 36.44Flow Rate: 1.0 mL/minλ: 280 nm
27512
0 1 2 3 4 5 6 7 8 9 10–0.0120
0.0120
AU
Minutes
Run #100
Run #80 Run #70 Run #60 Run #50 Run #40 Run #30 Run #20 Run #10
Run #90
Column: MAbPac SCX-10, 4 × 250 mmEluent: 20 mM MES, 200 mM NaCl, pH 6.0Flow Rate: 1.0 mL/minλ: 254 nmSample: Cyt C
27513
21.8
–0.0001
0.0180
AU
0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.00.0001
0.0168
AU
Minutes
Phosphate buffer
MES buffer
Lys
Cyt C
Rib
Lys
Cyt C
Rib
Column: 4 × 250 mmA: 20 mM MES, pH 6.0 B: 20 mM MES, 1.0 M NaCl, pH 6.0 t (min) %A %B 0.0 100 0 20.25 52 48
Column: 4 × 250 mmA: 10 mM Na2HPO4, pH 6.0 B: 10 mM Na2HPO4, 1.0 M NaCl, pH 6.0 t (min) %A %B 0.0 100 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nm
Flow Rate: 1.0 mL/minλ: 254 nm
9
The pH of the mobile phase is another parameter to be considered in any method development. In this case (Figure 14) the protein mix was tested in the column with the same MES buffer solution but at various pH values. The results confirmed that the selectivity of proteins can be adjusted by changing the pH of the mobile phase
Figure 14. Effect of pH of the MES buffer on the separation of the protein mixture.
conclusIons• A new MAbPac SCX column was developed for monoclonal antibody
separations. This is a complimentary addition to existent ProPac WCX columns providing high resolution and orthogonal selectivity for various proteins and MAb charge variant characterization.
• A controllable and robust surface grafting technique (ATRP-graft from) was applied that provided uniform functional layer for ion exchange.
• High resolution and high efficiency were obtained for monoclonal antibody variants.
• Ruggedness of the column was proved within broad pH and temperature ranges.
• Column reproducibility met industry standards.
ProPac and Chromeleon are registered trademarks of Dionex Corporation.
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27514
–0.0004
0.0129
AU
–0.0011
0.0170
AU
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22.6–0.0003
0.0127
AU
Minutes
pH 6.0
pH 6.4
pH 5.8
LysCyt C
Rib
LysCyt C
Rib
LysCyt C
Rib
Column: 4 × 250 mmA: 20 mM MES B: 20 mM MES, 1.0 M NaCl t (min) %A %B 0.0 100 0 20.25 52 48
Flow Rate: 1.0 mL/minλ: 254 nm