Post on 02-Nov-2019
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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)