separation of monoclonal antibodies by weak cation...

ABSTRACTMonoclonal antibodies (MAbs) are currently developed by pharmaceutical and biotechnology companies for various therapeutic applications. MAbs undergo several post-translational modifications including oxidations, deamidations, lysine truncations, and glycan modifications. Manufactur-ing of MAbs and subsequent stability testing procedures involve routine analysis and monitoring of the impurities resulting from asparagine deami-dation, aspartic acid isomerization, disulfide interchange, peptide bond cleavage, and oxidation. ProPac® WCX and ProSwift™ WCX columns are widely used to characterize MAb heterogeneity.

ProPac WCX columns are packed with particles that are well suited for analytical separations for monoclonal antibody variants. We have separated MAbs using various gradients, pH, and temperatures and have suggested optimal conditions for separation of monoclonal antibodies. We also suggested a conditioning step to recover under-performing columns. The conditioning is done by treating the column at 50 °C for 48 h in presence of 20 mM MES pH 5.6 (2-[N-Morpholino] ethanesul-fonic acid) or, alternatively, treating the column at 70 °C for 7 h. When MAbs are separated on these heat-conditioned columns, they exhibited restored efficiency, peak shapes, and improved overall performance as compared to unconditioned under performing control columns.

ProSwift monolith columns offer significant advantages over conven-tional porous columns, including fast mass transfer, high loading capacity, and wide pH stability. This exclusive combination of characteristics sup-ports versatile performance in a wide range of biomolecule separations, including monoclonal antibody separations. ProSwift WCX columns offer excellent capacity with high resolution, making them suitable for the first dimension of multi-dimensional chromatography.

INTRODUCTIONMonoclonal antibody (MAb) microheterogeneity can be attributed to glycosylation, oxidation, mutation, phosphorylation, amino terminal modifications (e.g., to pyroglutamate), incomplete processing of the C-terminus, and asparagine (Asn) deamidation. These variations in protein composition occur in many types of proteins and can impact their activity and stability of biotherapeutics. Monitoring stability of

therapeutic proteins and peptides is essential for demonstrating safety and efficacy of these drugs and is required by the FDA and other regula-tory agencies. These variations are routinely monitored preferably by cation-exchange chromatography.

Dionex cation-exchange ProPac WCX1-3 and ProSwift WCX columns are ideal for resolving closely related protein charge variants. ProPac pack-ings are pellicular polymeric supports with hydrophilic coatings and grafted surface chemistry which exhibit minimal hydrophobic character. In addition, these particles exhibit a wide range of pH stability with high selectivity and minimal band spreading. ProSwift monolithic columns are specifically designed to provide high-resolution and high-efficiency separations of proteins. ProSwift media are based on polymeric monoliths prepared by an in situ polymerization process. They are a new generation of separation media, which are uniquely designed and engi-neered for separation of biomolecules. The monolith is a single cylindri-cal polymer rod containing an uninterrupted, interconnected network of through pores, which are also called channels. The ProSwift columns have excellent resolution and also high capacity. They are available with reversed-phase and ion-exchange surface chemistries, and provide fast protein separations using conventional HPLC.

In this study we present data using ProPac WCX and ProSwift WCX col-umns. Excellent separations are achieved for the immunoglobulin G (IgG) MAb charge variants. We test the effect of pH, temperature, and NaCl gradients on MAb separation. The authors suggest a simple column conditioning step for ProPac WCX column to improve the resolution of MAbs. In this procedure, the column is heat treated in presence of 20 mM MES pH 5.6 or 6.5 (2-[N-Morpholino] ethanesulfonic acid) for 48 h at 50 °C or, for 7–8 h at 70 °C. When MAbs are separated on the heat conditioned columns, they exhibited higher efficiency, better peak shapes and improved overall performance as compared to separations performed on unconditioned control columns. The authors used the ProSwift WCX column for characterizing MAb heterogeneity. Due to high capacity of the ProSwift WCX, milligram quantities can be loaded on to the column for separation and fractionation of variants for further characterization. The authors compared the separation of MAb variants using different buffer components at various pH conditions. Also, we compared the ProSwift WCX column with a leading WCX biocolumn for MAb separations and present the results.

Srinivasa Rao, Andy Woodruff, Maria Rey, Kelly Flook, Jim Thayer, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USASrinivasa Rao, Andy Woodruff, Maria Rey, Kelly Flook, Jim Thayer, and Chris Pohl, Dionex Corporation, Sunnyvale, CA, USA

Separation of Monoclonal Antibodies by Weak Cation-Exchange Chromatography Using ProPac and ProSwift Columns

Separation of Monoclonal Antibodies by Weak Cation-Exchange Chromatography Using ProPac and ProSwift Columns

Anuta

New Stamp

2 Separation of Monoclonal Antibodies by Weak Cation-Exchange Chromatography Using ProPac and ProSwift Columns

Figure 1. Crystal structure of a human IgG1 MAb.

ProPac WCXMATeRIAlSChromatographic ComponentsMAb Separations: All PEEK™ System

ICS-3000 DP gradient pump, VWD absorbance detector, AS autosam-pler and TCC-10 thermostatted column compartment (Dionex Corporation)

Chromatography was controlled by Chromeleon® Chromatography Data System software (Dionex Corporation).

ChemicalsProtein Standards, MES, HEPES and all other analytical grade chemicals were obtained from Sigma. MAb is a gift from a biotech company.

ColumnsProPac WCX-10 (P/N 054993) analytical columns are from Dionex Corporation. These columns consist of a polymer support coated with a hydrophilic polymer.

ProSwift WCX-1S 4.6 × 50 mm (P/N 064295) and 1 × 50 mm (P/N 066643) are polymer monoliths from Dionex Corporation.

Competitor A: Leading weak cation-exchange column, 10 µm, 1000 Å, 5 × 50 mm

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)

25709

3Separation of Monoclonal Antibodies by Weak Cation-Exchange Chromatography Using ProPac and ProSwift Columns

Figure 2. ProPac WCX: phase design and mechanism of retention.

Figure 3. Identification of MAb variants. The humanized lgG1 MAb variants differing in heavy chain C-terminal lysine content were resolved using a shallow NaCl gradient. Differences in C-terminal lysine were verified by treatment of the MAb with carboxypeptidase B, an enzyme that cleaves C-terminal lysine residues.

Figure 5. Effect of temperature (20 °C to 60 °C) on MAb separation at pH 5.5.

Figure 4. Effect of pH on MAb separation. Panel A displays from pH 5.0 to 9.0. Panel B shows a zoomed version to point out variants separation from pH 5.0 to 8.0. Excellent separation was achieved at pH 5.5.

_+

+

OO OO- R

( ) n10 µm nonporouspolymeric substrate Highly crosslinked

hydrophilic layerwith reaction sites

Polymeric graftswith anionic sites

R = selectivity enhancing group

ProPac WCX

• 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 pH gradient

Cationic residue/siteon the protein

Anion-exchange sitecovalently bound to resin

OO

27688-01

0 5 10 15Minutes

A. Control MAb

B. MAb treated with carboxypeptidase B (2 h, 37 °C)

Column: ProPac WCX-10, 4 × 250 mmEluents: A) 10 mM sodium phosphate, pH 7 B) 1M NaCl, 10 mM sodium phosphate, pH 7Gradient: 4–15% B in 30 minFlow Rate: 1 mL/min Inj. Volume: 10 µLDetection: UV at 220 nmSample: Humanized IgG1 MAb AU

14063

K KK

0 52.0

0.040

AU

pH 9.0

pH 8.5

pH 8.0pH 7.5

pH 7.0pH 6.5pH 6.0 pH 5.5

pH 5.0

4.2 45.0-0.0021

0.0115

AU

pH8.0

pH7.5

pH7.0

pH6.5

pH6.0

pH5.5

pH5.0

Minutes

Column: ProPac WCX-10, 4 × 250 mmEluents: A) 20 mM HEPES + 20 mM MES + 20 mM TAPS B) 20 mM HEPES + 20 mM MES + 20 mM TAPS + 250 mM NaCl Gradient: Time (min) %A %B -10 95 5 0 95 5 50 5 95 52 5 95 52.5 95 5Temperature: 30 °CInj. Volume: 20 µLFlow Rate: 1.0 mL/minDetection: UV at 280 nmSample: 2.5 mg/mL MAb

A

B

23746

-0.005

Column: ProPac WCX-10 ,4 × 250 mmSystem IEluents: A) 20 mM MES B) 20 mM MES + 250 mM NaCl (NaCl Gradient 3.75 mM/min)Gradient: Time (min) %A %B 0 95 5 60 5 95 62 5 95 62.5 95 5Inj. Volume: 20 µLFlow Rate: 1.0 mL/minDetection: UV at 280 nmSample: 2.5 mg/mL MAb

0 60-0.01

0.05

AU

Minutes

20 °C

30 °C

40 °C

50 °C

60 °C

23747

Figure 6. Effect of salt gradients (4.75 mM/min to 2.25 mM/min) on MAb separation at pH 5.5.

Figure 7. Effect of MES/50 °C treatment on WCX-MAb separation. The ProPac WCX-10 column was treated with 20 mM MES pH 6.5 at 50 °C for 48 h. The treated column was used to separate MAb and compared with an untreated column. The chromatography was performed at 30 °C.

Column: ProPac WCX-10, 4 × 250 mmEluents: A) 20 mM MES B) 20 mM MES + 250 mM NaCl (NaCl Gradient 3.75 mM/min)Gradient: Time (min) %A %B 0 95 5 60 5 95 62 5 95 62.5 95 5Inj. Volume: 20 µLFlow Rate: 1.0 mL/minDetection: UV at 280 nmSample: 2.5 mg/mL MAb

0 52-0.01

0.06

AU

Minutes

4.75 mM/min

4.25 mM/min

3.75 mM/min

3.25 mM/min

2.75 mM/min2.25 mM/min

23748

22628

Column: ProPac WCX-10, 4 × 250 mmEluent: A) 20 mM MES, pH 6.5 B) 20 mM MES + 200 mM NaCl, pH 6.5Flow Rate: 1 mL/min Inj. Volume: 10 µLDetection: UV at 280 nmSample: MAb, 1 mg/mL

10

Chromatography was performed at 30 °C

20 30Minutes

40 50 600

90

mA

–20

35.0

75.0

0.0MES/50 °C treated for 48 h

Untreated


Figure 8. Dionex ProSwift family columns.

Figure 9. Morphology of ProSwift WCX-1S: SEM image.

ProSwift WCX

Figure 11. MAb separated on the ProSwift WCX-1S using different buffer conditions.

Figure 12. Assay of MAb variants (PTAD buffer) with and without carboxypeptidase.

Key: NewProducts

Product Line

1 × 50 mm

ProSwift RP-3U064298

ProSwift SAX-1S068459

ProSwift RP-10R164397

ProSwift RP-4H069477

4.6 × 50 mm 4.6 × 50 mm 1 × 50 mm


ProSwift RP-1S064297

ProSwift WAX-1S064294

ProSwift WAX-1S066642

ProSwift WCX-1S064295

ProSwift SCX-1S066765

ProSwift SCX-1S071977

ProSwift SAX-1S064293

CurrentProducts

Ion-Exchange

23874-05

1 × 250 mm

ProSwift WCX-1S066643


Reversed-Phase

ProSwift WCX-1S(Magnification 4000x)

24339

mAU

600

–1000 Minutes 14

Column: ProSwift WCX-1S, 4.6 × 50 mmEluents: A) 10 mM Sodium phosphate, pH 7.6 B) 1 M Sodium chloride in eluent AGradient: 5–10% B in 10 min, 100% B for 4 minFlow Rate: 1 mL/minInj. Volume: 10 µLTemperature: 30 °CDetection: UV at 214 nmSample: MAb 5 mg/mL

Acidic variants Basic variants

K

KK

Lysine truncationvariants

23839

Figure 10. Separation of MAb on ProSwift WCX-1S.

24564

Column: ProSwift WCX-1S, 4.6 × 50 mmFlow Rate: 1 mL/minInj. Volume: 8 µLTemperature: 30 °CDetection: UV at 280 nmSample: MAb, 5 mg/mL each

Eluents and MES, pH 6.5Gradient: 95–220 mM NaCl in 15 min

Tris, pH 7.5, 64–164 mM in 30 min

Sodium phosphate, pH 7.5, 40–140 mM in 30 min

Minutes 30

MES

Tris.HCl

NaHPO4

mAU

0

24565

Column: ProSwift WCX-1S, 1 × 50 mmEluents: A) 15 mM PTAD,* pH 6 B) 1 M NaCl in eluent AGradient: 4.5–18.8% B in 20 minFlow Rate: 200 µL/minInj. Volume: 10 µLTemperature: 30 °CDetection: UV at 215 nmSample: MAb ± carboxypeptidase

*PTAD: buffer mix containing equimolar phosphate, Tris, AMP and diisopropylamine

B is a magnified view of A.

mAU

Carboxypeptidase treated MAb

Native MAb

Acidic Variants

Basic Variants

Lys TruncationVariants

Carboxypeptidase Control

mAU

Minutes

Carboxypeptidasetreated MAb

Native MAb

Acidic Variants

Basic VariantsVariants not =related to Lys

Carboxypeptidase Control

0

A

B

24

Minutes0 24

0 1 2 3 4 5 6 7 8 9 10 11 12-100

0

100

200

300

400

500

600

mAU

280

nm

Minutes

0.223

0.208

0.2010.2090.215

1

23

10 µg20 µg

80 µg120 µg

40 µg

Basic variants

00.5 1 1.5 2 2.5

0.15

0.25

Log Sample Load (µg)

PW1/

2 (m

in)

Column: ProSwift WCX-1S, 1 × 50 mmEluents: A) 10 mM Sodium phosphate pH 7.6 B) 1 M NaCl in eluent AGradient: 1–15% B in 7.5 minFlow Rate: 0.2 mL/minInj. Volume: Variable 2–24 µLTemperature: 30 °CDetection: UV, 280 nmSample: MAb 5 mg/mL

Peak width at the half height (min) is shown for peak 3.


24349

Figure 13. Loading capacity of ProSwift WCX-1S.

5Separation of Monoclonal Antibodies by Weak Cation-Exchange Chromatography Using ProPac and ProSwift Columns

Figure 14. Assay of MAb variants with and without carboxypeptidase on WCX-1S.

Figure 15. Comparison of ProSwift WCX-1S with competitor A: separation of MAb.

Column: ProSwift WCX-1S, 4.6 × 50 mmEluents: A) 10 mM Tris, pH 7.6 B) 1 M NaCl in eluent AGradient: 5–10% B in 10 minFlow Rate: 1 mL/minInj. Volume: 16 µLDetection: UV at 280 nmSample: 1. MAb 720 µg (no carboxypeptidase B) 2. MAb 720 µg + carboxypeptidase (144:1)

mAU

Minutes 140

1

2

3

MAb

Lys Variants(1,2,3)

MAb + CP

24353

Minutes

-100

600

mAU

-100

600

mAU

Columns: ProSwift WCX, 4.6 × 50 mm, Competitor A, 5 × 50 mm Eluents: A) 10 mM Sodium phosphate, pH 7.6 B) 1 M NaCl in eluent AFlow Rate: 1 mL/minInj. Volume: 10 µLDetection: UV at 214 nmSample: MAb 5 mg/mL

Gradient Flow Rate: 1 mL/min 5–10% B in 10 min Gradient Flow Rate: 2 mL/min 5–10% B in 5 minCompetitor A

1 mL/min

ProSwift WCX1 mL/min

0 5 10 14

0 5

?

10 14

0 5 10 14-50

500

mAUProSwift WCX

2 mL/min



24496

Column: ProSwift WCX-1S, 1 × 50 mmEluents: A) 10 mM Sodium phosphate, pH 7.6 B) 1 M NaCl in eluent AGradient: 2–83% B in 7.5 minFlow Rate: 0.2 mL/minInj. Volume: 0.5 µLTemperature: 30 °CDetection: UV at 214 nmSample: Protein mix, 1 mg/mLPeaks: 1. Ribonuclease A 2. Cytochrome C 3. Lysozyme

mAU

Minutes0 10

After 1 M NaOH wash for 1 hr

Before

24566

ProSwift WCX— RUggeDNeSS AND STABIlITy

Figure 16. Ruggedness of ProSwift WCX-1S.

Figure 17. ProSwift WCX-1S: Base stability.

0

mAU

Minutes1 2 3 4 5 6 7 8

790

170

251337

421

13

Inj #

Column: ProSwift WCX-1S, 4.6 × 50 mmEluents: A) 10 mM Sodium phosphate, pH 7.6 B) 1 M NaCl in eluent AGradient: 0–100% B in 5 minFlow Rate: 2 mL/minInj. Volume: 10 µLDetection: UV at 214 nmSample: Protein mix, 1 mg/mLPeaks: 1. Ribonuclease A 2. Cytochrome C 3. Lysozyme

2

24352


Chromeleon and ProPac are registered trademarks and ProSwift is a trademark of Dionex Corporation.

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CONClUSIONThe ProPac WCX-10 column is used routinely for separation of acidic and basic MAb variants, papain digested MAb components, and for other protein applications.

The authors have studied effect of pH, temperature, and different salt gradients on MAb separation using ProPac WCX-10 column. All these parameters appear to influence MAb separation and therefore play an important role in method development.

The authors have suggested a simple conditioning step to improve and obtain the maximum MAb resolution: treating the column at 50 °C for 48 h in a 20 mM MES buffer at pH 5.6 or 6.5 or, alternatively, treating the column at 70–80 °C for 7–8 h. When MAbs are separated on these MES/heat conditioned columns, they exhibited higher efficiency, better peak shapes, and improved overall performance compared to uncon-ditioned control columns. Once the column is heat treated, there is no need to repeat this step for subsequent runs. The authors assume that this change is permanent, although the authors have not yet evaluated how long this change may last. The authors hypothesize that change in the conformation of the polymer grafts may be responsible for this improved performance.

ProSwift media are based on polymeric monoliths prepared by an in situ polymerization process. They are a new generation of separation media, uniquely designed and engineered for fast separation of biomolecules.

ProSwift monolith columns offer significant advantages over conven-tional porous columns. They include fast mass transfer, high loading capacity, and wide pH stability. These exclusive characteristics support versatile performance in a wide range of biomolecule separations including monoclonal antibodies.

ProSwift WCX columns are available in different dimensions: 4.6 × 50 mm and 1.0 × 50 mm. Columns with 1 mm i.d. offer improved sensitivity and reduced solvent consumption.

ProSwift WCX columns are rugged and offer high capacity with excellent resolution making them suitable to be used in the first dimension of multidimensional chromatography.

ReFeReNCeS1. Dionex Application Notes 125, 127, and 128.

2. Harris, R. J., et al J. Chromatogr B, 2001, 752: 233–245.

3. Moorhouse K. G., et al J. Pharm. and Biomed Anal. 1997, 16: 593–603.

separation of monoclonal antibodies by weak cation...

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