Over the past decade, there have been a growing number of mAb candidates entering the clinical pipeline. This results in a large increase on the demand for analytical characterization. This seminar discusses advances in analytical method development with analytical run times below 10 minutes for all routine methods with intelligent, integrated chromatography workflows. Orbitrap technology has been established as the most powerful MS technology for protein characterization. How this can be incorporated into a complete workflow for bio-pharma analysis is also discussed.

1. Trends and Developments in MAb
Screening and CharacterizationScreening and Characterization
Ken Cook
2014
MAb Characterization

2. Pharmaceuticals and Biopharmaceuticals
pharmaceuticals
Produced by chemical synthesis
200 - 2,000 daltons
1 - 5 reactive groups
Relatively stable
MAb
150,000 Da
Precisely defined chemical
entities
biopharmaceuticalsp
Genetically engineered
Produced in living cells
2,000 - 2,000,000 daltons
10 2000 reactive groups
 Biosimilar
10 - 2000 reactive groups
Moderately to highly labile
Complex; a mixture of closely
related variants
Aspirin
180 Da
Because of their complexity, it is not
possible to make identical copies of
biologic drugs. These products are
therefore referred to as “biosimilar”
rather than generic drugs. Developers
Biopharmaceuticals present unique analytical challenges.
We have unique analytical capabilities that address these challenges
g g p
seek to achieve “similarity” and
“comparability”.
We have unique analytical capabilities that address these challenges.

3. The “Complexity” Challenge in MAb Analytics
Steven Kozlowski, FDA, WCBP2010

4. Market Trends in Biopharma
• Greater productivity needed in
method development
I i d l t i li• Increasing development pipeline
for monoclonal antibody (MAb)
therapeutics
• Advances in automation in
upstream processes such as cell
culture and purification process
d l tdevelopment

5. Types of Biopharmaceuticals
Source: PhRMA 2013 Biologics Overview
 MAbs are the fastest growing class of drugs
 “more than half of biopharmaceuticals in development are antibodies”
 by 2016, 6 of the top 10 drugs will be MAbs
“…more than 700 biosimilars/biobetters in the development pipeline…”

6. Requirements for Biopharma Method Development
• Easy method development
• Fast in optimization
• Rapid and simple method
• Short runtimesShort runtimes
• Easy to set-up and to keep running
• Generic approach
• Instrument speed up options• Instrument speed up options
• Easy method transfer to QA/QCy

7. Regulatory Requirements
Protein Analytical Chemistry Techniques Used in the Testing of Biological Products
Protein Property Characterization Batch Release/Stability Further Development of Assay
Size / Aggregates Mass spec (intact mass), HPLC SDS-PAGE, SEC Impurity (aggregates, fragments)
Charge CE-IEF, IEC, pH-IEC CE-IEF, IEC, pH-IEC
Acylation, deamidation, sialylation
variants
tid i h d h bi i t ti
Hydrophobicity
peptide mapping, hydrophobic interaction
chromatography (HIC)
Deamidation, oxidation, (U)HPLC
Concentration Amino acid analysis, HPLC method, ELISA UV A280
LC/MS fl t l b li h id HPAE PAD (IC)
Carbohydrate analysis
LC/MS, fluorescent labeling, monosaccharide
composition
HPAE-PAD (IC)
(U)HPLC
Heterogeneity
2°, 3° Structure Circular dichroism, peptide mapping Disulphide mapping
Peptide Mapping LC/MS N C sequencingPeptide Mapping LC/MS, N- C- sequencing
AAA analysis (U)HPLC-FLD or (U)HPLC-CAD
Binding activity ELISA, Biacore ELISA, Biacore
P t C ll b d C ll b d tPotency Cell-based assays Cell-based potency assay
Identity Western blotting, peptide mapping, (U)HPLC
Western blotting, peptide
mapping,
Adapted from Camille Dycke et. al., GEN October 15, 2010Adapted from Camille Dycke et. al., GEN October 15, 2010

8. Topics
• Speeding Up HPLC MAb Characterization Analysis
LC Column Selectivity Developments in Column Chemistry for Mab• LC Column Selectivity – Developments in Column Chemistry for Mab
Analysis
• Reducing Method Development Time
• High Throughput & Automation Strategies
• Parallel LC Configurations and Multi-Step Automation
I t ti M S i t MAb A l i W kfl• Integrating Mass Spec into MAb Analysis Workflows
• 2D LC – MS Workflows
• Current Trends and Developments in Glycan Analysisp y y
• Novel Column Chemistry for HPLC Glycan Analysis
• Comparison of LC-based methods

9. Approaches to Faster LC Separations
• Faster separations can be achieved by…
(A) Compressed gradients (e.g. in IEC)
• Can speed up the separation; usually some loss of resolution
(B) Shorter columns
• Resolution compromised but often “good enough”
(C) Smaller particle size resins
S d th ti d ith t l f l ti• Speed up the separation, and without loss of resolution
(D) Combinations of the above( )

10. The Thermo ScientificBio RS System - What is New?
LPG-3400RS/HPG-3x00RS/DGP-3600RS
- NEW biocompatible 1034 bar (15,000 psi)
pump fluidics
WPS-3000TBRS
- NEW biocompatible in-line split-loop
(flow-through) 1034 bar (15,000 psi)
autosampler
TCC-3000RS/SD
- NEW biocompatible 1034 bar 2-pos, 6-port
and 10-port, and 6-pos, 7-port valves
Viper Fingertight Fitting Systemp g g g y
- NEW biocompatible 1250 bar (18,130 psi)
capillaries

11. Added Bioanalytical Capabilities
• pH and Conductivity Monitoring
• Used in protein purification and analysis
• Highest accuracy through temperature compensation of
conductivity and pH results
• Useful tool for pH gradient analysis in IEC

12. pH Difficulties With Phosphate Buffers and Blending
10.00
11.00 8 pH2 #17 0 pH
100.0
%C: 0.0 %
9.00 8
7
80.0
10% A
0% A
7.00
8.00
7
6
5
4
3 40% A
20% A
10% A
6.00
60% A
50% A
4.00
5.00
2
1 %B: 0 0 % 0 0
100% A
80% A
0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 28.0
3.00 min
1
Flow: 150 µl/min
%B: 0.0 % 0.0

13. Calibration of Protein A Titre with new Protein A column

14. Faster Separations Without Loss of Resolution with
Smaller Particle Size Resin
• Faster MAb charge variant analysis…by reducing column length,
gradient time & particle sizegradient time & particle size
10 0
16.0
A 10 μm, 4x250 mm MAbPac SCX
5.0
10.0
2
0.0 10.0 20.0 30.0 40.0 50.0
58.0mAU
20 0
30.0
6
8
B 3 μm, 4x50 mm MAbPac SCX
10.0
20.0
3
4
5
8
11
9
10
7
Minutes
0.0 5.0 10.0
15.0
-5.0
0.0
3
12
13
14
10
1
2
15
16
Minutes

15. HIC for MAb Analysis

16. ProPac HIC – Key Application
• Methionine oxidation monitoring
Column: Thermo Scientific™ ProPac ™Column: Thermo Scientific ProPac
HIC-10, 4.6 x 100 mm
Eluent: A. 1M (NH4)2 SO4 in 0.1 M
NaH2PO4,
80 Main MAb
Peak
pH 7.0
B. 0.1 M NaH2PO4, pH 7.0
Flow Rate: 0.75 mL/min
Met Oxidation
Inj. Volume: 100 µL (50 µg)
Detection: 220 nm
Sample: MAb
Peak
mAU
5 10 15 20 25
0
Minutes
0
Minutes

17. Characterization of Aspartic Acid Variants
Valliere-Douglas, et al. (2008) J Chrom. A 1214, 81-89

18. Speed up of Mab Aggregate Analysis - SEC
55.0
60.0
1 - Gel Filtration dionex 15cm #18 MAb + Caffeine UV_VIS_1
2 - GEL FILTRATION DIONEX #16 [normalized] Caffiene UV_VIS_1
mAU
1 - 2.471
WVL:214 nm
40 0
45.0
50.0
30cm column15cm column
Aggregation
analysis in under 4
30.0
35.0
40.0
MAb
analysis in under 4
minutes
15 0
20.0
25.0
dimer
5.0
10.0
15.0
Caffeine
-10.0
-5.0
0.0
min
21
Less than 4 minutes!
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0

19. New Multi-Product Method
pH Gradient IEC

20. Ion Exchange Protein Elution Mechanisms
Isoelectric
Point (pI)
+
Buffer pH typically < pI
 Cation-Exchange
NH3
R +
COO-
Cationic protein
binds to
negatively charged
cation exchanger
+ ++
++
0
Buffer/System pH
 Cation Exchange
Chromatography
NH3
R +
COOH
+ ++
3 4 5 6 7 8 9 10
Buffer pH typically > pI
 Anion-Exchange
Chromatography
2
Anionic protein
binds to
positively charged
anion exchanger
- -
- -
-
–
Chromatography
NH2
R
COO-
- -
-
Protein net charge vs. pH

21. Mechanism of Salt and pH Elution of Proteins

22. Improving pH Gradient Cation-exchange Chromatography
of mAbs by Controlling Ionic Strength
Journal of Chromatography A, 1272 (2013) 56– 64

23. Buffer Development Strategy
• Replace cationic buffer components with zwitterionic buffer species
(Good’s Buffers)
• These buffer species contain one quaternary amine group and one sulfonic
acid group. They do not bind to the stationary phase in the
pH range of 6-10.p g
• They are not repelled by the stationary phase so they can buffer the stationary
phase.
MES MOPS TAPS CAPSO
6.1 7.2 8.4 9.6

24. Linear pH Gradient
Programmed gradient vs measured pH
Cytochrome C
y = 1.6923x - 7.2914
R² = 0.9929
9.5
10
10.5
ue
Protein pI vs. measured pH at elution
y = 0.1548x + 5.0404
R² = 0.9996
9 5
10.5
Programmed gradient vs. measured pH
Trypsinogen
Ribonuclease A
7 5
8
8.5
9
suredpHvalu
8.5
9.5
edpHvalue
L ti 1
Lectin - 2
Lectin - 3
Trypsinogen
6
6.5
7
7.5
Meas
Measured pH value
Linear (Measured pH Value)
6.5
7.5
Measure
Measured pH
Linear (Measured pH)
Lectin - 1
5.5
7.5 8.5 9.5 10.5
pI value
60.0
55
3
5.5
0 10 20 30 40
Retention Time [min]
Linear (Measured pH)
30.0
40.0
50.0
nce[mAU]
tin-1-5.87-6.04
-6.20
18-6.37
Trypsinogen-15.97-7.5
leaseA-22.00-8.53
tochromeC-31.55-9.93
10.0
20.0
Absorban
Lect
Lectin-2-6.97
Lectin-3-8.1
Ribonucl
Cy
0 5 10 15 20 25 30 35 40
-5.0
Retention Time [min]

25. Programmed Gradient and Actual Monitored pH
10.00
10.50 Novartis Method #3 Sample 1 pH
100.0
%C: 0.0 %
%D: 0.0 %
9.00
9.50
1 pH unit
i 5 i
8.00
8.50
in 5 minElution points for the
same protein 20
minutes apart with the
same programed
7.50
sa e p og a ed
gradient!
6.50
7.00
Origonal Method
6.00
Flow: 1.000 ml/min
%B: 0.0 % 0.0
g
Thermofisher Buffers
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0
5.20 min

26. Example #2: Herceptin, 5mg/mL,
MabPac SCX-10, 10µm 4x250 mm
15.0
30.0
5.0
10.0
0.0
i
%B: 10.0
Salt gradient
0.0 5.0 10.0 15.0 20.0 25.0 30.0 min
15.0
50.0
5 0
10.0
0.0
5.0
%B: 25.0
25.0
pH gradient
30 min gradient, MabPac SCX-10, 10 µm, 4x250 mm
0.0 5.0 10.0 15.0 20.0 25.0 30.0
min

27. 4-Protein Standards –
Thermo Scientific CX-1 pH Gradient Buffer
1
2
90
10.00
11.00
2
Lectin-1 - 6.15 - 6.11
60
9.00
(mAU)
pH trace
20
40
7.00
8.00
Absorbance
Ribonuclease A - 22 38 - 8 72
Cytochrome C - 31.88 - 10.15
20
6.00
1
Lectin-2 - 7.25 - 6.28
Lectin-3 - 8.45 - 6.45
Trypsinogen - 16.41 - 7.75
Ribonuclease A - 22.38 - 8.72
2
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
-10 5.00
Retention Time (min)

28. 4-protein Standards – PO4 Based pH Gradient
1 - 20130626_MABPACSCX4x250_001664_agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC UV_VIS_1
2 20130626 MABPACSCX4x250 001664 agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC pH
10 µm, 4 x250 mm
60.0
70.0
9.50
10.00
2 - 20130626_MABPACSCX4x250_001664_agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC pH
mAU
µ ,
40.0
50.0
8 00
8.50
9.00
pH trace
5 µm 4 x150 mm
20.0
30.0
7 00
7.50
8.00pH trace
5 µm, 4 x150 mm
0.0
10.0
6.00
6.50
7.00
1
2
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
-10.0 5.50
min

29. MAb Charge Variant Separation, 0–100% B
100% B0% B
40.0 10.50
30.0
9.00
mAU]
pH trace(a)
20.0
7 00
8.00
bsorbance[m
10.0
6.00
7.00
Ab
0 5 10 15 20 25 30 35 40
-5.0 5.00
Retention Time [min]
*The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and ConductivityThe pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity
Monitoring Module (PCM-3000)

30. MAb Charge Variant Separation, 25–50% B
25% B 50% B
16.0 8.00
10 0
7.75
mAU]
(c) pH trace
5.0
10.0
7.25
7.50
bsorbance[m
5.0
7.00
Ab
0 5 10 15 20 25 30 35 40
-2.0 6.60
Retention Time [min]

31. Protein Loading with a Salt Gradient
2,000 p _ _
mAU WVL:280 nm
1 600
1,800
mAU WVL:280 nm
80.0
%C: 0.0 %
MAbPac SCX 4 x 250mm
1,400
1,600
1,000
1,200
Peak Width
600
800
Resolution
200
400
1 2mg
0
3
2
1
Flow: 1000 µl/min
%B: 33.3 %
1.2mg
0.3mg
0.1mg
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
-300 min

32. Protein Loading with a pH Gradient on the Same Column
1,000 3 pH Buffer B test #18 Cap6 UV_VIS_1
900
1,000
mAU WVL:280 nm
100.0
%C: 0.0 %
MAbPac SCX 4 x 250mm
700
800
500
600
Peak Width
400
500
Resolution
200
300
%B: 40.0 %
100
3
2
1
1 - 12.328
2 - 21.105
Flow: 1000 µl/min
25.0
1.2mg
0.3mg
0.1mg
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0
-20 min

33. Effect of Column Length on pH Gradients
The resolution is surprisingly
similar even when the column
length is changed
dramatically. The main
difference is the elution time
which can be attributed to thewhich can be attributed to the
higher capacity of the long
column.
This is suggesting that thegg g
primary mechanism of
separation is the pH gradient
itself and the effect on the PI
of the proteinof the protein.

34. Fast Runs-Protein Standards: 20 Min Run vs 10 Min Run
70.0
1 - 2013-10-01_MPSCX-10_5um_sn001050 #2 LTRC, 3:2:3:2, pH calibrated UV_VIS_1
mAU WVL:280 nm
Az
Flow rate at 1 mL/min, 15min gradient/ 20 min totally cycle time
40.0
20.0
1
-10.0
60.0
2 - 2013-10-01_MPSCX-10_5um_sn001050 #4 LTRC, 3:2:3:2, pH calibrated UV_VIS_1
mAU WVL:280 nm
Flow rate at 2 mL/min, 7.5min gradient/ 10 min totally cycle time
12 5
25.0
37.5
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
-10.0
12.5
min
2
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

35. Herceptin, 0-100% B
140 11 00
_ _ _ p , g p
120
140
10.00
11.00
mAU
100
9.00
10.00
60
80
8.00
9.00
40
7.00
20
6.00
-20
0
5.00
min
1
2
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

36. Herceptin, 25-50%B
40 0 8 20
AU
35.0
40.0
8.00
8.20
mAU
25 0
30.0
7.80
20.0
25.0
7 40
7.60
10 0
15.0
7.20
7.40
5.0
10.0
7.00
-5 0
0.0
6 60
6.80
min
1
2
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
-5.0 6.60

37. Different mAb2 Using Fast pH Gradient
138
160 PH gradient_Oct2013 #32 mAb UV_VIS_1
mAU
1 - 4.014
WVL:280 nm
100.0
%C: 0.0 %
As fast as CE Analysis!
113
125
As fast as CE Analysis!
75
88
100
50
63
55.0
13
25
38
2 - 4.268
0
13
Flow: 450 µl/min
%B: 32.0 % 32.0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
-20 min

38. 0 to 100% Start for a 10 Minute pH Gradient
mAU WVL:280 nm
500 9
9 different Mab samples
375 8
125
250 7
6
0 5
4
-250
-125
4
3
500
-375
min
2
1
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
-500 min

39. Summary
• pH Gradient IEC is perfectly suited as a platform method
• Allowing generic methods for multi-product analysis
• Even with pI ranges from 5 – 10
• Simple and fast method development
• pI value of the unknown MAb can be predicted from the correlation curvepI value of the unknown MAb can be predicted from the correlation curve.
• Easy optimization of the method
• Less dependant on sample matrix and sample preparation
• High loading capacity for low level analysis as well as
variant fractionationvariant fractionation
• Fast high resolution methods using short columns
• High capacity methods for fractionation using longer columns
• Robust

40. High-Throughput and Automation Strategies
• Tandem and Parallel LC Configurations
• To increase sample throughput of validated methods• To increase sample throughput of validated methods
• Multi-step Automation
• To automated multi-step workflow e.g. MAb purification and analysis on a
single LC platform
• Reduce hands-on timeReduce hands on time
• Case Studies
• Fast MAb Aggregate Analysis
• Automated MAb Titer Threshold Method

41. Parallel LC for Dual Assays Aggregate and Variants
Both AnalysisBoth Analysis
with one
injection in 10
Minutes!
IEC SEC
Minutes!
IEC SEC
Increases throughput, eliminates the need to duplicate sample plates

42. System Configuration
A B C
DGPDGP
A B CDual gradient pump
‘Two LPG pumps in a single unit’
(upgradeable with solvent selection valves)
DGPRightDGPLeft
Fraction collecting autosamplerColumn oven with
UV WPS
g p
‘inject – collect – re-inject’column selection valves
up to 6 or 10
columns / positions Injection
collectionUV WPS collection
Waste
Prot A
SEC
Prot A
IEC

43. Typical mAb Workflow
A B C
DGPDGP
A B C
Sample loading onto protein A
DGPRightDGPLeft
UV
Injection
collection
WasteWPSUV WPS
Prot A
LOAD + WASH
SEC
Prot A
IEC

44. Typical mAb Workflow
A B C
DGPDGP
A B C
Elution and fractionation
DGPRightDGPLeft
UV
Injection
collection
WasteWPSUV WPS
Prot A
ELUTE + FRACTIONATE
SEC
Prot A
IEC

45. Typical mAb Workflow
A B C
DGPDGP
A B C
Second dimension SEC analysis
ion
s
on
s
DGPRightDGPLeft
80
100
125
mAU
UV214nm
UV280nm
Agglomerat
products
PI
Degradatio
products
UV
Injection
collection
WasteWPS
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
-10
20
40
60
min
21
AP
UV WPS
Prot A
SEC
Prot A
IEC

46. Typical mAb Workflow
A B C
DGPDGP
A B C
Second dimension IEC analysis
DGPRightDGPLeft
20.0
30.0
mAU
K
K K
UV
Injection
collection
WasteWPS
0.0 20.0 40.0 60.0 80.0
0.0
10.0
min
Acidic Variants
Basic Variants
UV WPS
Prot A
SEC
Prot A
IEC

47. Key Columns for Biopharma Analytics
Analysis Description Columns
MAb Capture &
Titer Analysis
Mab capture for analysis workflows; Mab
titer determination (concentration) &
screening
MAbPac Protein A  “The gold standard in
antibody analysis”
Charge Variant
Analysis
routine charge variant profiling/screening;
including lysine truncation, acylation &
deamidation; done by CEX & AEX
ProPac WCX-10
MAbPac SCX-10
MAbPac SCX-10RS
CX-1 pH Gradient Buffer Kit
ProPac SAX-10
Pro-Pac WAX-10  Robust, multi-product , high
resolution pH gradient IEC
 superior resolution for most
MAb samples tested
Aggregate
Analysis
routine screening for Mab aggregates and
fragments
MAbPac SEC-1
Glycan Profiling profiling of released glycans Accucore Amide-HILIC
resolution pH gradient IEC
 Novel GlycanPac column
chemistry separates glycans byy g p g g y
GlycanPac AXH-1
GlycanPac AXR-1
Intact Protein &
Subunit Profiling
ADC DAR analysis; glycoform profiling;
LC/HC and Fab/Fc analysis; disulfide
ProSwift RP-10R
ProSwift RP-2H&4H
size, polarity and charge. Mass
Spec compatible.
ProSwift RP-10R monolithic
Subunit Profiling LC/HC and Fab/Fc analysis; disulfide
mapping
ProSwift RP 2H&4H
Accucore 150-C4
MAbPac SEC-1
Sequence &
Structural
Analysis
primary sequence analysis; peptide
mapping; peptide & glycopeptide
structural & linkage analysis
Acclaim PepMap
PepSwift (PS-DVB)
Acclaim RSLC 120, C18
column provides highest
resolution and lowest
carryover for intact MAb mass
analysis.
Analysis g y ,
Accucore 150-C18
Trp Oxidation &
Deamidation; ADC
analysis
targeted analysis of tryptophan oxidation
& deamidation
ProPac HIC-10
MAbPac HIC-10
 ProPac HIC – novel chemistry
for Trp oxidation; orthogonal to IEC
and SEC for variant analysis.

48. Integrating MS into mAb Analysis
Workflows

49. Seamless Integration of Salt-Based SEC, IEC, HIC Methods
to MS for Characterization of MAb Products and Impurities
S l A l i
Exact Mass Determination, Bottom-up,
and Top-Down Protein CharacterizationAutomated Bio LC-LC/MS
1-D LC
ProA, SEC, IEC or HIC Data Analysis
Deconvolution of ESI-MS
Sample Analysis
Using HR/AM Mass
Spectrometers
Fraction Collection of MAb
Products or Impurities
to zero charge accurate
mass
80
90
100
2997.31777
2920.44812
2664.49830
Products or Impurities
[Automated in Autosampler]
z=?
2000 2500 3000 3500
m/z
0
10
20
30
40
50
60
70
RelativeAbundance
2178.40685
3535.70427
3619.47065
Automated 2-D LC
SPE/Desalting on RP
followed by MS 1311.0 1311.5 1312.0 1312.5
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
z ?
1311.87212
R=70792
z=?
1311.54737
R=68130
z=?
1311.98174
R=69867
z=?
1311.43967
R=58977
z=? 1312.09147
R=67084
z=?
1312.20087
R=56981
z=?
1311.31799
R=88597
z=?
1310.98824
R=45346
z=?
1312.42258
R=47666
z=?
1312.64646
R=43558
z=?
23565 23570 23575 23580 23585 23590
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
23578.58636
23580.66451
followed by MS m/z m/z

50. System Configuration
A B C
DGPDGP
A B CDual gradient pump
‘Two LPG pumps in a single unit’
(upgradeable with solvent selection valves)
DGPRightDGPLeft
Fraction collecting autosamplerColumn oven with
UV WPS
g p
‘inject – collect – re-inject’column selection valves
up to 6 or 10
columns / positions Injection
collectionUV WPS collection
Waste
RP
SEC
RP
IEC

51. MAb IEX Fraction Desalting using Monolithic Columns
with Consecutive Blanks
120
1 - RP MAB #17 MAb UV_VIS_1
2 - RP MAB #19 blank UV_VIS_1
3 - RP MAB #20 blank UV_VIS_1
mAU WVL:280 nm
%C: 0.0 %
90
100
110 90.0
%C: 0.0 %
60
70
80
30
40
50
1 - 5.093
10
20
30
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
-20
-10
0
min
321
Flow : 250 µl/min
%B: 10.0 % 10.0

52. Accurate MW Determination of Reduced IgG Light Chain
80
85
90
95
100
1303.0917
z=18
1465.9155
z=16
IgG light chain
18+ charge state
45
50
55
60
65
70
75
veAbundance
1234.6137
z=19 1563.5760
z=15
1675.1174
z=14
240,000 resolution
10
15
20
25
30
35
40
45
Relativ
1172.8326
z=20
1803.8945
z=13
1954.1323
z=12 2131.7785
z=11
1117.1264
z=21
240,000 resolution
1200 1400 1600 1800 2000 2200 2400
m/z
0
5
10
1302.6 1303.0 1303.4 1303.8
m/z
Xtract
d l tideconvolution
Measured mass = 23424.4845
Target mass = 23428.416g
4 Dalton Mass Deviation  2 S-S?
How do we confirm this?How do we confirm this?
Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University

53. pH Gradient Separation of Purified IgG on a MAbPac
SCX-10 Column

54. Deconvoluted Results from MS Spectra

55. mAb Peptide Map – Normal / Stressed Sample
350
200
mAb normal
0
100
2
1
-100
mAb Stressed
-200
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0
-350
min
mAb digest normal and stressed, 5 mg/mL, 30 min gradient
Acclaim C18 2.2µm 2.1x250mm SN 1041

56. Asparagine to IsoAspartic Acid Detection
N+1 shift

57. Current Trends & New
Developments in Glycan Analysis

58. LC-MS Analysis of Labeled Glycan: HILIC Amide Column
100
19
20
Commercial amide HILIC column (1.7 µm)
ce
15
18
14 &17
16 15
eAbundanc
14
11a &c
12b, 13 &16
12b
13
Relativ
6,7 & 10
15
21 & 22
23
11a &c
1010
1 2 4
5
9
12a & 13
25
24
7
26
6 6 or 7
0 10 20 30 40 50 60
Minutes
0
1 2 9
Conventional HILIC columns do not separate by charge; glycans co eluteConventional HILIC columns do not separate by charge; glycans co-elute

59. LC-MS Analysis of Labeled Glycan: GlycanPac AXH-1
100
14
nce
12 b
20
12a
12b
veAbundan
11a-c
12a-b
15
19
Relativ
5
13
0
1
2 3
4
5
6
7
8
9
10
18 21
22
23
24
25 26
16
17
P k d i t l “ l t ” ith th h
0 10 20 30 40Minutes
0
Mono- Di- Tri - Tetra-Neutral
Peaks grouped into several “clusters” with the same charge

60. 16E6
Charge-based Separation for Easy Quantitative Analysis
16E6
3
4
P k Gl T
Relative
Peak Glycan Type
%
1 Neutral 0.4
2 Mono-Sialic 8 6
ceCounts
2 Mono Sialic 8.6
3 Di-Sialic 38.4
4 Tri-Sialic 45.4
2
5
Fluorescenc
5 Tetra-Sialic 7.0
6 Penta-Sialic 0.2
1
5
6
7.00 8.00 9.00 10.00 11.00 12.00
Minutes
Quantitative Determination of each glycan charge state

61. Separation of 2AA Labeled N-glycans from IgG by GlycanPac
AXH-1 (1.9 µm) Column: pH 5.1 in 25 oC
Column: Thermo Scientific ™ GlycanPac™
AXH-1 (1.9 µm)
Dimension: 2.1x150 mm
1.8E6
3
Mobile phase: A: acetonitrile
B: water
C: ammonium Acetate (100 mM, pH =5.1)
Flow: 0.4 mL/min
3
8
Flow: 0.4 mL/min
Temp: 30 oC
Injection: 5 pmoles
Detection: fluorescence detector
Sample: 2AB Labeled N glycan from IgG
13
enceCounts
Sample: 2AB Labeled N-glycan from IgG
Time
(min)
% A % B C%
Flow
Rate
(mL/
min)
-10 81 18 1 0.4
9
17
Fluoresce
0 81 18 1 0.4
25 74 18 8 0.4
35 62 18 20 0.4
0
1 2 4
5
6
7
10
11
12
14 15
16
10.0 20.0 30.0
0
Minutes

62. Separation of N-glycan by Thermo Scientific™ Acclaim® Glycan A
XR Column 2.1x150mm, 1.9 um
700,000
counts
Time
(min)
% A %B %D Flow
(mL/min)
0 0 5 95 0.4
20 4 18 78 0 4
Eluent: A: Acetonitrile B: Ammonium formate (0.1M,
pH = 4.4) D; water
0.4 mL/min
20 4 18 78 0.4
24 0.7 30 69.3 0.4
44 6 30 64 0.4
60 15 30 55 0.4
44 numbers of peaks
Peaks width is better than
3 l3um column
-100 000
-50,000
0
min
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0
-100,000

63. Comparison of the Three New Glycan Columns
Glycan HILIC WAX 1.9um
Glycan WAX – RP 1.9um

64. GlycanPac AXH-1, AXR-1
• High resolution columns for separation and structural characterization
of biologically relevant glycansof biologically relevant glycans
• UHPLC column suitable for high-throughput analysis
• UHPLC-FLD for fluorescently labeled N-glycans
• LC-MS and LC-MS/MS for structural characterization of both
labeled and native N- and O-glycans from proteins by MS detection

65. Trend Toward HR/AM MS for Intact IgG Mass
Measurement e.g. Glycoform Analysis
80
85
90
95
100
2745.7720
2851.3544
75
80
85
90
95
100
2745.7720
2797.5697
2695.8919
R: 17.5K
40
45
50
55
60
65
70
75
elativeAbundance
2907.25952556.4844
2965.3764
2471.3405
3025 8632
2391 6288
25
30
35
40
45
50
55
60
65
70
RelativeAbundance
5
10
15
20
25
30
35
40
Re
3025.8632
2391.6288
2680 2700 2720 2740 2760 2780 2800 2820
m/z
0
5
10
15
20
25
1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000
m/z
0
-7 ppm
G0F+G1F
-0 7 ppm 8 5
G0F+G0F
G0F+G2F (or 2G1F)
0.7 ppm -8.5 ppm
-0.9 ppm
Protein
Deconvolution 1.0
G1F+G2F
G2F+G2FG0+G0F
G0+G02xMan5
5.0 ppm
0.9 ppm
Q Exactive
O bit MS
G1F+G2F+SA
In-depth characterization, comparability studies e.g. process
change; originator vs. biosimilars comparability
Orbitrap MS

66. Summary
• Unique construction of ProPac ion-exchange phases enables high resolution separations
of protein isoforms and other closely-related protein variants
• Protein A column for rapid capture and Titre of IgG
• The ProPac HIC has improved hydrolytic stability compared to other silica-based HIC
columns with better resolution than polymer-based HIC columns
• The ProPac SEC column enables high performance protein separations by size for
aggregate anal sis in less than 4 min tesaggregate analysis in less than 4 minutes
• Dual analysis can be carried out with short runs using different chemistries
• ProSwift monolithic RP columns useful for fast high resolution separations of large
proteins with ultra low carryoverproteins with ultra low carryover.
• GlycanPac columns for unique separation and resolution of Glycans
• Bio-Compatible inert Viper connections for ultra low dispersion
The U3000 BioRS system allow biocompatible Mab UHPLC analysis and automated 2• The U3000 BioRS system allow biocompatible Mab UHPLC analysis and automated 2
dimensional capture and analysis steps. Doing the work of multiple instruments in one.

67. Thank You—Q&A
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