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INTRODUCTION

Therapeutic monoclonal antibodies often

contain low-level variants that alter protein

surface charge, and can significant impact

mAb function and efficacy. These charge

variants can result from modifications

including: C-terminal lysine retention,

deamidation, oxidation, and glycan sialylation.

Characterization of these modifications

presents an analytical challenge due to overall

sample heterogeneity, and the low abundance

of most charge variants. We have applied

protein Ion Exchange Chromatography (IEX)

for the isolation and enrichment of mAb charge

variants of Infliximab, and LC/MSE peptide

mapping for characterization of isolated acidic

charge variant peaks.

Robert Birdsall, Vera Ivleva, Ying Qing Yu, Scott J. Berger and Weibin Chen Waters Corporation 34 Maple Street, Milford MA, 01757

RESULTS & DISCUSSION

Instrumentation

Waters ACQUITY® H-Class Bio System with 2D Technology

Waters Xevo G2-S Q-Tof MS

Columns

Protein-Pak Hi Res SP Column,7µm, 4.6 x 100 mm (SCX)

ACQUITY UPLC Protein BEH C4 Column,

300Å, 1.7 µm, 2.1 mm X 50 mm

ACQUITY UPLC Peptide BEH C18 Column,

130Å, 1.7 µm, 2.1 x 100 mm

IEX (1st dimension)

Mobile Phase:

A: 100 mM MES monohydrate

B: 100 mM MES sodium salt

C: 1000 mM NaCl

D: 18 MΩ water

Gradient:

25-65 mM NaCl in 15 minutes

RPLC (2nd dimension)

Mobile Phase:

A: 18 MΩ water, 0.1% FA, v/v

B: Acetonitrile, 0.1% FA, v/v

Gradient:

0-95 %B in 10 min

Temp: 80 °C

MS (peptide):

Capillary: 3kV

Sample cone: 80 V

Source temp: 120 °C

Desolvation temp: 300 °C

RPLC

Mobile Phase:

A: 18 MΩ water

B: Acetonitrile

C: Acetonitrile, 1 % FA, v/v

Gradient:

13-52% Acetonitrile, 0.1%

FA, v/v in 60 min

Temp: 65 °C

CONCLUSIONS

On-line Enrichment of Low Abundance mAb Charge Variants using ACQUITY H-Class Bio with 2D Technology

No. of cuts

trapped

1

2

4

3

5

Retention time [min]

2nd Dimension (RPLC)

Enrichment results

Figure 3. On-Line Enrichment. The highly linear response of protein recovery versus the number of heart-cut replicates analyses (Figure 2) indicates a reproducible method for the on-line enrichment, desalting, and elution of

lower abundance protein variants.

AUTOMATING CHARACTERIZATION OF mAb CHARGE VARIANTS ISOLATED BY CATION EXCHANGE CHROMATOGRAPHY USING AN INTEGRATED LC/MS PEPTIDE MAPPING AND INFORMATICS WORKFLOW

An automated peptide mapping workflow enabled us to localize the modifications contributing to the change of surface charge, streamline the process of da-ta collection, processing, and review. Comparative tabular and chromatographic tools enabled us to highlight dif-

ferences and trends across multiple samples.

Fraction B (acidic peak)

Fraction F (main peak) peptide T7

deamidated T7

METHODS

Sequence Coverage Map

Fraction A

Fraction B

Fraction D

Fraction E

Fraction F T7 peptide

% Modification of Identified

Deamidated Components

Charge Variant Characterization Using UNIFI Peptide Mapping Workflow

88 % sequence coverage was obtained in

the heavy chain peptide map

T7 peptide fragment [SINSATHYAESVK]

exhibited a characteristic shift in retention time associated with a deamidation event

T7 acidic variant

T7 main peak

L-isoaspartyl

form

L-aspartyl

form

1 Da shift on starting on y10 ion

*

*

MSE

1 2

3

1

2

3

Figure 9. Charge Variant Modification MSE Analysis. The Deamidation of asparagine undergoes modification from asparagine to a succinimide intermediate that degrades into isoaspartic acid and aspartic acid (~ 3:1 ratio) as seen in the left panel. From the automated workflow, MSE analysis of the peptides identified a 1 Da shift in the isoapartic and aspartic acid containing deamidated peaks, evident in the y-ions associated with the acidic variant T7 peptide.

This study demonstrated an efficient automated workflow that successfully:

Enabled differences in charge variant profiles to be determined

across samples

Localized and identified

modifications giving rise to individual charge variant peaks

Streamlined analysis and reporting of results from multiple complex data sets

Figure 7. Component XIC Profile Analysis. The T7 peptide was compared across 5 enriched IEX fractions. The pair of later eluting peaks in fraction A and B are char-acteristic of deamidation. Fraction B had the highest level of deamidated T7 pep-tide.

Figure 8. Relative Quantification of Modification. The relative amount of T7 deamidation was approximately 30% for IEX fraction B and largely absent in the maps of other digested fractions. LCMSE mapping was performed in triplicate for each digested IEX fraction.

Component Profile Analysis

Figure 6. Mirror Plot Comparison. The mirror mode comparative chromatographic view allows rapid visualization of variant peptides against a control map. For this specific example, the T7 peptide of acidic variant fraction B was identified as containing a novel deamidated peak, readily observed when compared against the peptide digest of the main peak (fraction F) of the monoclonal antibody.

Quantification of Modifications

Figure 5. Streamlining Data Review with Workflows. Data review workflows were defined that configured display elements and applied data filters to enable focus on specific scientific questions. As shown in the top panel, complex data sets can be probed for specific modifications, then switched to an alternative view for con-

firmation of assignment quality using MSE fragmentation data (bottom panel).

“one-c

lick” w

ork

flow

Bioinformatic Search for “Acidic” PTM’s

Position 2: A heart-cut is performed on Peak 2, and desalted

using the BSM pump

Position 1: Desalted fractions can be directed for MS analysis

or collected for enzymatic digestion.

AS

1st dimension (IEX)

2nd dimension (RPLC)

TUV/PDA

TUV/PDA

BSM

Xevo

G2S

QSM AS

1st dimension (IEX)

2nd dimension (RPLC)

TUV/PDA

TUV/PDA

Xevo

G2S

QSM AS

1st dimension (IEX)

2nd dimension (RPLC)

TUV/PDA

TUV/PDA

Xevo

G2S

.

. .

.

.

. .

.

waste waste Peak 2

Peak 1

Peak 3

Flow

position

1 position

1

position

2 position

2

position

1 position

1

. waste

Peak 1

Peak 2 Fraction trapped on column head

Peak 3

Flow

.

unbound salt

Peak 1 & 3

unbound salt

Flow

Position 1: Sample is injected on and resolved by the 1st

dimension IEX column.

Peak 2 fraction

ACQUITY H-Class Bio with 2D Technology

Valve Configuration

QSM

BSM

BSM

B A D

F CPB treated

Infiximab

Retention time [min]

1st Dimension (IEX)

C

Figure 2. Low Abundance Charge Variants of Infliximab. Inflixmab sample was treated with carboxypeptidase B to reduce sample complexity and increase representation of acidic fraction peaks (Peaks A—E). Peak B was enriched

on-line (multiple runs) to minimize protein loss typical of

offline fraction collection.

heart-cut

E acidic variants

Figure 1. 2DLC Valve Configuration. Tandem column configurations (e.g. IEX/RPLC) with synchronized valve switching were used to “heart-cut” selected peaks from the 1st dimension IEX column to the 2nd dimension RP desalting column.

A heart-cut (Figure 2) onto

a 2nd dimension can be used

to enrich low abundance

species (peak A-E) on-line

for increased productivity

and sample recovery. The

2nd dimension column acts

as a trapping/desalting

column to retain and enrich

multiple 1st dimension

heart-cuts (Figure 3).

Figure 4. Method Processing Setup. As part of the integrated acquisition/processing method, the set of most common modifications leading to acidic protein variants was selected for the bioinformatic search. This included sialic acid containing glycans, and PTMs such as deamidation and oxidation.

The ACQUITY® H-Class Bio with

2D Technology supported the on-line

fractionation, desalting, and enrichment of

low abundance charge variant peaks.

A B D E F

*axis off-set for contrast

Informatics:

with

2D Technology

LCMSE Peptide Mapping Analysis was automated using the Biopharmaceutical Platform Solution with UNIFI®

Deamidated

form

Confirming Charge Variant Modification Using MSE Fragmentation Data

B

Simplifying data review with predefined

workflow steps (defined views/filters)