Monoclonal Antibody Heterogeneity Characterization Using...

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Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns Srinivasa Rao, Yuanxue Hou, Yury Agroskin, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USA Srinivasa Rao, Yuanxue Hou, Yury Agroskin, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USA ABSTRACT Monoclonal 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. INTRODUCTION Monoclonal 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.

Transcript of 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.

Anuta
New Stamp
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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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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

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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

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70

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mAU

Minutes

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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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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

Minutes

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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

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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

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Column C

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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.

North America

U.S./Canada (847) 295-7500 South America

Brazil (55) 11 3731 5140

europe

Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722

Asia pacific

Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190Taiwan (886) 2 8751 6655

Dionex Corporation

1228 Titan Way P.O. Box 3603 Sunnyvale, CA 94088-3603 (408) 737-0700 www.dionex.com

LPN 2708-01 12/10©2010 Dionex Corporation

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.