monoclonal antibody heterogeneity characterization using...
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Monoclonal AntibodyHeterogeneity Characterization Using Cation-Exchange Columns
Monoclonal AntibodyHeterogeneity Characterization Using Cation-Exchange ColumnsSrinivasa Rao, Yuanxue Hou, Yury Agroskin, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USASrinivasa Rao, Yuanxue Hou, Yury Agroskin, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USA
ABSTRACTMonoclonal antibodies (MAbs) have been approved to treat vari-ous diseases, such as cardiovascular and inflammatory diseases and cancer, among others. More than 20 MAbs have been approved, and an additional 150 MAbs are currently in preclinical or clinical trials, or awaiting FDA approval. In the last decade, the MAbs market has grown exponentially, resulting in multibillion dollar annual sales revenue. Ac-cordingly, therapeutic proteins and monoclonal antibodies now form the largest part of the growing biologics drug market and have transformed the biotechnology and biopharmaceutical industries.
Monoclonal antibodies generally exhibit charge heterogeneity from oxida-tion, asparagine deamidation, aspartic isomerization, lysine truncation, glycan modifications, and other modifications. Therefore, the manufacture and testing procedures of MAbs involve routine analyses and monitor-ing of impurities resulting from these situations. Weak cation-exchange (WCX) columns are well suited and widely used for the characterization of MAb heterogeneity. ProPac® WCX columns packed with nonporous 10 μm particles with carboxylic acid functional groups are well suited for high-resolution analytical separations of target MAbs from both acidic and basic variants. The authors recently developed a new, strong cation-exchange (SCX) column for MAb analysis with sulfonate functionality and an alternative selectivity, that is complementary to the ProPac WCX column.1
The new SCX column is built with a newly designed hydrophilic phase with a well-controlled, uniform, and stable ion-exchange separation surface on highly crosslinked polymeric nonporous resin. Here, the authors demonstrate heterogeneity characterization of MAbs using both the ProPac WCX and the new SCX columns, emphasizing the utility and applications of this new SCX column. Further, reproducibility and robustness of the new column are also demonstrated.
INTRODUCTIONMonoclonal antibodies (MW 150 kDa) are composed of one heavy chain (MW 50 kDa) and two types of light chains (kappa and lambda, MW 25 kDa).2 They generally exhibit charge heterogeneity from oxidation, asparagine deamidation, aspartic isomerization, lysine truncation, glycan modifications, and other modifications. Therefore, manufacturing and subsequent quality assurance and stability testing procedures of MAbs involve routine analyses and monitoring of the impurities resulting from these situations. Ion-exchange columns (IEX), in general, and weak cation-exchange (WCX) columns, in particular, are well suited for the characterization of MAb heterogeneity.
ProPac weak cation-exchange (WCX) columns are routinely used to characterize MAb heterogeneity as they are well suited for high-resolution analytical separations of both acidic and basic monoclonal antibody vari-ants. ProPac packings are pellicular polymeric supports with hydrophilic coatings and grafted surface chemistry, which exhibit minimal hydro-phobic character. In addition, these particles exhibit a wide range of pH stability with high selectivity and minimal band broadening.
Here, the authors discuss the latest development of a new MAb column—the MAbPac™ SCX-10—that is designed as a complementary addition to the ProPac WCX column, providing high resolution and orthogonal selectivity for various proteins and MAb charge variant characterization. Using this column, several applications including characterization of lysine truncation variants, Fab, and Fc fragment analysis are presented. Also, lot-to-lot and column-to-column reproducibility and ruggedness of the new MAbPac column are demonstrated.
2 Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns
Figure 1. Crystal structure of a human IgG1 MAb.
Figure 2. Separation media and mechanism of cation-exchange columns.
K K
Heav
y cha
in
Papain cleavage site
Fe
Asparagine (red)deamidation and
glycosylation variants
Aspartic acid (orange)isomerization variants
Methionine (light blue)oxidation variants
Cysteine (green)disulphide and
oxidation/reducingvariants
Serine (yellow)glycosylation variants
Histidine (blue)oxidation variants(metal-catalyzed)
Threonine (purple)glycosylation variants
E.A. Padlan: "The Protein Data Bank", Mol. Immunology, 1994, 31, 169.
Interchaindisulphide bonds
Intrachaindisulphide bonds
Light chain
Light
chainConstant
VariableVariableF ab
antige
n bind
ing sit
e
Fab'
antigen binding site
Heavy chain
Diagram of a IgG1 Monoclonal Antibody (MAb)
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MATeRIAlSChromatographic Components MAb Separations
A PEEK™/Inert system including: ICS-3000 DP gradient pump, VWD absorbance detector, UltiMate® Autosampler and thermostatted column compartment (TCC-100) from Dionex Corporation.
Chromeleon® Chromatography Data System software (Dionex Corporation).
Chemicals, MAbMES, Tris, and all other analytical grade chemicals were obtained from Sigma. MAb was a gift from a local biotech company.
Columns MAbPac SCX-10, 4 × 250 mm (P/N 074625) and ProPac WCX-10, 4 × 250 mm (P/N 054993) columns from Dionex Corporation.
SepARATION MeDIA AND MeChANISMSubstrate10 μm nonporous, highly crosslinked styrene-type polymeric media was used.
ProPac WCX Column1. A proprietary surface modification process to create a highly
hydrophilic coating. No hydrophobic interactions are present.2. Random polymerization approach used for functional grafts.3. Functional group: carboxylic acid.
MAbPac SCX Column1. A proprietary, different one-step surface modification process
yielding uniform hydrophilic coating. No hydrophobic interactions are present.
2. ATRP-based grafting approach to control functional group chain length and density of functional groups.
3. Functional group: sulfonic acid.
Cationic residue/site on the protein
Anion-exchange site covalently bound to resin
-+
+
• Mechanism of retention: charge-charge interaction• Separation based on total charge of protein• Highly dependent on ionic strength• Highly dependent on pH• Elution by increasing ionic strength, or by pH gradient
Hydrophilic Layer
Grafted Ion-Exchange Layer
Highly CrosslinkedPolymetic Micro55 Bead
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3Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns
SeleCTIVITY DIFFeReNCeS
Figure 3. Selectivity differences: MAbPac SCX-10 vs ProPac WCX-10 columns. Ribonuclease A elutes prior to other proteins in MAbPac SCX-10 as compared to ProPac WCX-10 showing a selectivity change. Such orthogonal selectivity differences are quite helpful in protein heterogeneity characterization.
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 mmEluents: A) 20 mM MES, pH = 6.4 B) 20 mM MES, 1.0 M NaCl, pH = 6.4 Time (min) %A %B 0.0 100 0 20.25 52 48
Flow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: UV at 254 nmSample: 1. Ribonuclease A (Rib) 1 mg/mL 2. Cytochrome C (Cyt C) 0.2 mg/mL 3. Lysozyme (Lys) 0.25 mg/mL
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propac WCX-10 vs MAbpac SCX-10 COlUMN
Figure 4. MAb separation: Comparison of ProPac WCX-10 vs MAbPac SCX-10 columns. The ProPac WCX-10 and MAbPac SCX-10 columns provide high resolution and peak efficiencies of monoclonal antibody acidic, basic, and C-terminal lysine variants.
–2
18
0 10 20 30 40 50 58–2
16
A
B
1
4 3 2
5
6
8
7
11
9 10 12 13 14 15 16 17
2 5
6
8
7
11
10 12 15 16 17
3 4 1
9 14 13
Gradients: A: 25–46.44% B in 50 min B: 15–36.44% B in 50 min Flow Rate: 1 mL/minTemperature: 30 °CInj Volume: 5 µL Detection: UV at 280 nmSample: MAb, 10 mg/mLPeaks: 1–5. Acidic variants 6, 8, 11. C-Terminal lys variants 12–17. Basic variants
Columns: A) ProPac WCX-10, 4 × 250 mm B) MAbPac SCX-10, 4 × 250 mm Eluents: A) 20 mM MES + 60 mM NaCl pH 5.6 B) 20 mM MES + 300 mM NaCl, pH 5.6
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ApplICATIONS OF The MAbpac SCX-10 COlUMN
Figure 5. MAb separation on the MAbPac SCX-10 column using different gradi-ents. MAb analysis was achieved under 15 min with high resolution.
Figure 6. pH gradient-based MAb separation.3 With the use of a pH gradient, the MAbPac SCX-10 column provides high-resolution separation of monoclonal antibody variants.
0 5 10 15 18
0
70
0 5 10 15 20 25 28
0
30
0 5 10 15 20 25 30 35 40 45 48
0
20
A
B
C
mAU
Minutes
mAU
mAU
Column: MAbPac SCX-10, 4 × 250 mm Eluents: A) 20 mM MES + 60 mM NaCl pH 5.6 B) 20 mM MES + 300 mM NaCl, pH 5.6 Gradient: A) 15–40% B in 10 min B) 15–40% B in 20 min C) 15–40% B in 40 min
Flow Rate: 1 mL/minTemperature: 30 °CInj Volume: 10 µLDetection: UV at 280 nmSample: MAb, 5 mg/mL
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27691
15 20 30 40 45
2
22
1 2 3 4 5
6
7
9
10
11
12 13 14
8
3525
Column: ProPac MAb SCX, 4 × 250 mm Eluents: A) 2.4 mM Tris + 1.5 mM imidazole + 11.6 mM piperazine pH 6.0 B) 2.4 mM Tris + 1.5 mM imidazole + 11.6 mM piperazine pH 10.5Gradient: 40–100% B in 60 minFlow Rate: 1 mL/minTemperature: 30 °CInj. Volume: 10 µLDetection: UV at 280 nmSample: MAb, 5 mg/mLPeaks: 1–7. Acidic variants 8, 9, 11. C-terminal lys variants 12–14. Basic variants
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4 Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns
Figure 7. Analysis of MAb lysine truncation variants on the MAbPac SCX-10 column. A second chromatogram (B) verifies that the three major peaks are due to variations in the presence of C-terminal lysine. After carboxypeptidase B treatment, C-terminus lysine is removed and only one major peak remains.
Figure 8. Analysis of Fab and Fc fragments of a MAb after digestion with car-boxypeptidase and papain using the MAbPac SCX-10 column.
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Column: MAbPac SCX-10, 4 × 250 mmEluents: A) 20 mM MES, pH 5.6 + 60 mM NaCl B) 20 mM MES, pH 5.6 + 300 mM NaClGradient: 15–36.44% B in 50 minFlow Rate: 1 mL/minTemperature: 30 °CInj. Volume: 5 µLDetection: UV at 280 nm
Samples: A. MAb, 900 µg in 100 µL (no carboxypeptidase) B. MAb, 900 µg in 100 µL + carboxypeptidase, 50 µg, incubation at 37 °C for 3 hBoth Chromatograms: Peaks 1–5. Acidic variantsSample A: Peaks 6–8. C-Terminal lysine truncation variants of main peak Peaks 9–11. C-Terminal lysine truncation variants of minor variant peakSample B: Peak 6 results from peaks 6, 7, and 8 after CBP treatment Peak 9 results from peaks 9, 10, and 11 after CBP treatment
mAU
Minutes0 10 20 30 40 50 58
0
27.5
A
B
11 10 9
8 7
6
5 4
6
5 4
3 2 1
3 2 1 9
0 10 20 30 40 50 58
0
40
G
F
E
D
C
B
A
1
2 3 4
5
6 7
8 9
10
11
1
2 3
5
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8 9 4 7
10 11 12
13
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Column: MAbPac SCX-10, 4 × 250 mm Eluents: A) 20 mM MES + 60 mM NaCl, pH 5.6 B) 20 mM MES + 300 mM NaCl, pH 5.6 Gradient: 1–35% B in 50 min Flow Rate: 1 mL/minTemperature: 30 °CInj Volume: 5 µLDetection: UV at 280 nmTotal Volume: 300 µLSamples: A. Carboxypeptidase blank 10 µL (50 µg; no MAb) B. Papain blank 10 µL (100 µg; no MAb) C. Carboxypeptidase 50 µg + papain 100 µg D. MAb 3 mg/300 µL + papain 10 µL (100 µg) E. MAb 3 mg/300 µL + papain 10 µL (100 µg) + carboxypeptidase 10 µL (50 µg) F. MAb 3 mg/300 µL G. MAb 3 mg/300 µL + carboxypeptidase 10 µL (50 µg)
Sample D, papain treatedPeaks: 1–4. Acidic variants 5, 6, 7. C-Terminal lys truncation variants of main peak 13–14. Fab PeaksSample E, papain and carboxypeptidase treatedPeaks: 1–4. Acidic variants. Peak 5 results from peaks 5, 6, 7 after papain and CPB treatment 10–11. Fab peaks
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lOT-TO-lOT-RepRODUCIBIlITY
Figure 9. Lot-to-lot reproducibility of the MAbPac SCX-10 column. Columns from each of the three different lots (A, B, C) were used for MAb analysis. Reten-tion times for the C-terminal lysine variants are shown.
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–0.00027
0.00549
AU
1 - 25.224
2 - 28.124
3 - 32.147
–0.00006
0.00492
AU
1 - 24.497
2 - 27.297
3 - 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 - 28.074
3 - 32.090
A
B
C
Column: MAbPac SCX-10, 4 × 250 mmEluents: A) 20 mM MES, 60 mM NaCl, pH = 5.5 B) 20 mM MES, 300 mM NaCl, pH = 5.5 Time (min) %A %B 2.0 85 15 52.0 63.56 36.44
Flow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: UV at 280 nmSample: MAb 5 mg/mL
COlUMN-TO-COlUMN RepRODUCIBIlITY
Figure 10. Column-to-column reproducibility of the MAbPac SCX-10 column. No change in performance was noted for 4 different MAbPac SCX-10 columns (A, B, C, D) from the same lot.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56–0.0043
0.0084
AU
Minutes
Column D
Column C
Column B
Column A
Column: MAbPac SCX-10, 4 × 250 mmEluents: A) 20 mM MES, 60 mM NaCl, pH = 5.5 B) 20 mM MES, 300 mM NaCl, pH = 5.5 Time (min) %A %B 2.0 85 15 52.0 63.56 36.44Flow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: UV at 280 nmSample: MAb 5 mg/mL
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5Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns
MAbPac is a trademark and Chromeleon, ProPac, and UltiMate are registered trademarks of Dionex Corporation.
PEEK is a trademark of Victrex PLC.
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RUGGeDNeSS OF The MAbpac SCX COlUMN
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/minDetection: UV at 254 nmInj. Volume: 10 µL Sample: Cytochrome C 0.125 mg/mL
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Figure 11. Ruggedness of the MAbPac SCX-10 column. More than 100 isocratic runs were performed to assess the reproducibility, ruggedness, and column performance. The chromatography profiles remain unchanged.
CONClUSIONS• A new MAbPac SCX column was introduced for monoclonal
antibody separations. This is a complementary addition to existent ProPac WCX columns, providing high resolution and orthogonal selectivity for various proteins and MAb charge variant characterization.
• A controllable and robust ATRP surface grafting technique was ap-plied that provided a uniform functional layer for the ion-exchange process.
• High resolution and high efficiency were obtained for monoclonal antibody variants analysis as well as various MAb fragmentation analysis.
• Columns exhibited lot-to-lot and column-to-column reproducibility.• Ruggedness of the column was proved within broad pH and tem-
perature ranges.
ReFeReNCeS1. MAbPac SCX-10 manual and data sheets available at
www.dionex.com.2. Bennett, K. L.; Smith, S. V.; Truscott, R. J. W.; Sheil, M. M.
Anal. Biochem. 1997, 245, 17–27.3. Farnan, D.; Moreno, G. T. Anal. Chem. 2009, 81, 8846–8857.