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Biopharmaceutical Platform Solution with UNIFI 1.7
Biopharmaceutical Platform Solution
Intact Protein Mass
Peptide Mapping
DDA (Peptide & Glycan)
Xevo G2-S QTof
©2012 Waters Corporation 2
Bioseparations
Size Exclusion (UV)
Glycan)
Released
Glycan
GU + Mass
Intact Protein: TUV, MSPeptide Mapping: TUV, MSE, MS/MSReleased Glycan: FLR (+MS, NIBRT Library), MS/MS Bioseparations: TUV, FLR
Workstation or Workgroup (Compliance)
UNIFI system based
Workflow and Solutions for
©2012 Waters Corporation 3
Characterization of mAbs
Characterisation Workflow – Core Requirements
ReductionAlkylationDigestion
Host Cell
Variant Profiling
Higher Order Structure
UPLC/ MSE
Peptide Map
Intact (mAb) Mass by LC/MS
©2012 Waters Corporation 4
Reduction PNGaseF DeglycosylationHILIC Glycan SPE2AB Label & CleanupHILIC-FLR (MS)
Stability and Formulation
Host Cell Proteins
Bioanalysis
QC Testing
Released Glycan (FL/ MS)
Light Chain, Heavy Chain UPLC/ MS
Reduced LCMass Analysis
IntactMass Analysis
ReducedPeptide Mapping
Aggregate Analysis
Glycan Analysis
Non-ReducedPeptide Mapping
Analysis Workflow for mAbs
©2012 Waters Corporation 5
Reduced HCMass Analysis
Charge variantAnalysis
Reduced Deglyco HCMass Analysis
Analysis Workflow for mAbs
Aggregate Analysis
Glycan Analysis
Non-ReducedPeptide Mapping
IntactMass Analysis
Reduced LCMass Analysis
ReducedPeptide Mapping
©2012 Waters Corporation 6
Peptide Mapping
Charge variantAnalysis
Mass Analysis
Reduced Deglyco HCMass Analysis
Reduced HCMass Analysis
UPLC SEC Analysis for Intact Protein Aggregation
IgG monomer
IgG dimer0.15
0.20
0.25
Separates proteins by their size in solution (Stokes radius)
Separations are Isocratic
No concentration of analyte on the column
All analytes elute within 1 column volume
©2012 Waters Corporation 7
IgG dimer
AU
0.00
0.05
0.10
0.15
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Higher order
aggregates
ACQUITY BEH200, 1.7 µm, 4.6 x 150mm
Monitoring variable levels of aggregation among different batches
Monomer
Batches fromdifferent process
©2012 Waters Corporation 8
� ACQUITY UPLC-BEH200 SEC, 1.7 µm, 4.6 x 150mm Column (p/n=186004930), Temp: 25 C
� Conditions: 0.3 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl
Dimer% Dimer
I II III
Batch-to-Batch Comparison of Aggregation
©2012 Waters Corporation 9
Monomer : 99.3 to 99.5%
Dimer : 0.5 to 0.7%
Analysis Workflow for mAbs
AggregateAnalysis
Glycan Analysis
Non-ReducedPeptide Mapping
IntactMass Analysis
Reduced LCMass Analysis
ReducedPeptide Mapping
©2012 Waters Corporation 10
Charge variantAnalysis
AnalysisPeptide MappingMass Analysis
Reduced Deglyco HCMass Analysis
Reduced HCMass Analysis
Screening molecule Use as release assay
pH 6.825–200 mM
pH 6.825–100 mM
Optimizing an Ion-Exchange Intact mAbs Separation
pI is 8.3We run IEX at 6.8We expect protein to have net positive charge
©2012 Waters Corporation 11
pH 6.825–65 mM
Ionic S
trength
(m
M)
Retention Time (min)
Optimized Gradient
H2O
100mM Na2HPO4
100mM NaH2PO4
1000mM NaCl
Auto•Blend Plus™ Technology
Column: Protein-Pak Hi Res CM 4.6 x 100 mm
Optimizing an Ion-Exchange Intact mAbs Separation
pH
pH 6.28–6.6760 mM NaCl
0K
1K
2K
pH Gradient Salt Gradient
Ionic S
trength
(m
M)
0K
1K
2K
38–68 mMpH 6.8
©2012 Waters Corporation 12
AB+ allows the flexibility to use either pH or salt gradients when characterizing biomolecules
Retention Time (min)
pH
Ionic S
trength
(m
M)
Retention Time (min)
1K
H2O
100mM Na2HPO4
100mM NaH2PO4
1000mM NaCl
Column: Protein-Pak Hi Res CM 4.6 x 100 mm
IEX of Antibody (Intact)
0K
1K2K
©2012 Waters Corporation 13
Acidic Variants
Basic Variants
� Column: Protein-Pak HiRes Sp 7um 4.6 x 100 mm, Temp: 25 C
� Conditions: 0.5 mL/min; 20mM Sodium Phosphate, pH 6.8, Gradient: 0-50 mM NaCl in 10 min
Batch-to-Batch Comparison: Acidic variants across samples
©2012 Waters Corporation 14
AP1: 0.3 to 0.8% ; AP2: 0.1 to 0.4%; AP3: 0.2 to 1.2% ; AP4: 1.6 to 3.7%
AP5: 1.4 to 4.8%
Reduced LCMass Analysis
IntactMass Analysis
ReducedPeptide Mapping
Aggregate Analysis
Glycan Analysis
Non-ReducedPeptide Mapping
Analysis Workflow for mAbs
©2012 Waters Corporation 15
Reduced HCMass Analysis
Charge variantAnalysis
Reduced Deglyco HCMass Analysis
The conformation of the primary structures is the cornerstone in
the verification of the identity of therapeutic mAbs.
Lysine variants (0-2) are present in both samples with varying relative abundance
LC Mass spectra
PNGase F
KK KKDeglycosylated
MaxEnt1 Deconvolutedmass spectra 0K
1K
2K
©2012 Waters Corporation 16
Relative abundance of C-terminal lysine variants can also be compared by intact mass analysis
Batch-to-Batch Consistency%
No Lysine I II III
©2012 Waters Corporation 17
Good analytical reproducibility reveals lysine variant differences between batches
Sample Injections
% N
o Lysine
Orthogonal methods for multi-batch comparison (0K-Lysine Variants)
% A
mount
IEX-UV Intact
©2012 Waters Corporation 18
Sample Injections
Good analytical
reproducibility reveals
consistent results between
the analytical methods for
lysine variant differences%
Am
ount
LC/MS IntactDeglycosylated
KK
Antibody light chain mass suggests no Lys variation exist on the subunit
+
Reduction
LC HC
Reduced Mass Analysis of Antibody
MaxEnt1 Deconvoluted mass spectra Mass spectra
KKK
23434.0
LC Masse Match √No evidence of minor species √
©2012 Waters Corporation 19ACQUITY UPLC PrST BEH C4, 300A, 1.7 µm, 2.1 x 50 mm
InnovatorLC
MaxEnt1 Deconvoluted mass spectra Mass spectra
Deglycosylated Reduced Mass Analysis
+Reduction
LC HC
K
PNGase F
K KKK
1K
Antibody heavy chain (deglycosylated) mass suggests Lys variation exist on the subunit
©2012 Waters Corporation 20
0K
IntactMass Analysis
ReducedPeptide Mapping
Aggregate Analysis
Glycan Analysis
Non-ReducedPeptide Mapping
Analysis Workflow for mAbs
Reduced LCMass Analysis
©2012 Waters Corporation 21
Charge variantAnalysis
Reduced Deglyco HCMass Analysis
Mass Analysis
Reduced HCMass Analysis
Mass analysis of Antibody heavy chain reveals the heterogeneity of the subunit
MaxEnt1 Deconvoluted mass spectra Mass spectra
+
Reduction
LC HC
KKK
Innovator
LC
G0F +K
©2012 Waters Corporation 22
G0F
G0+K
Man5
G1F +K
G2F +K
Analysis Workflow for mAbs
ReducedPeptide Mapping
IntactMass Analysis
Aggregate Analysis
Glycan Analysis
Non-ReducedPeptide Mapping
Reduced LCMass Analysis
©2012 Waters Corporation 23
Charge variantAnalysis
Mass Analysis
Reduced Deglyco HCMass Analysis
Reduced HCMass Analysis
Experimental Setup for Peptide Mapping
Antibody
Denature & Alkylate
Trypsin DigestLC/MSE
Non-ReducedPeptide Map
Reduction &
Alkylation LC/MSE
Reduced Peptide Map
©2012 Waters Corporation 24
UPLC BEH300 C18, 1.7 µm, 2.1 x 100 mm
UNIFI Scientific Information System
UPLC/MSE Comprehensively Analyzes Complex Samples
� UPLC/MSE is a simple method of unbiased data acquisition that
comprehensively analyzes all components in a single analysis.
©2012 Waters Corporation 25
Protein coverage was obtained
HC Protein Coverage
©2012 Waters Corporation 26
LC Protein Coverage
BEH, c18, 1.7, 130, 2.1x 100 mm, 0.05%TFA, Gradient: 1 to 35% ACN, 60 min
Asp Isomerization of Peptide T24 (FNWYVDGVEVHNAK)
XIC
IsoASP
Isomerization: Asp to iso-Asp (no mass difference).isoAsp is not a natural amino acid and can potentially
©2012 Waters Corporation 27
isoAsp is not a natural amino acid and can potentially be immunogenic.
Oxidation of HC Peptide T42
II
©2012 Waters Corporation 28
% O
xidation
Sample Injections
I
Analysis Workflow for mAbs
IntactMass Analysis
Aggregate Analysis
Glycan Analysis
Reduced LCMass Analysis
Non-ReducedPeptide Mapping
ReducedPeptide Mapping
©2012 Waters Corporation 29
Charge variantAnalysis
Mass Analysis
Reduced Deglyco HCMass Analysis
Reduced HCMass Analysis
Peptide Mapping
Experimental setup for disulfide bond mapping
Antibody
Denature & Alkylate
Trypsin DigestLC/MSE
Non-ReducedPeptide Map
Reduction &
Alkylation LC/MSE
Reduced Peptide Map
©2012 Waters Corporation 30
UPLC BEH300 C18, 1.7 µm, 2.1 x 150 mm
UNIFI Scientific Information System
� 6 S-S bonds (12 intra, and 4 inter)
� Digestion Enzyme: Trypsin
� Symmetry of IgG1 molecule provides
redundancy in mass-based search
�8 unique S-S bonded peptides
Heavy chain
VH
CH1
C L
VL
Light chain
S– -–S
S– -–S
S– -–S
S– -–S
S–-–S
S–-–S
S–-–S
S–-–S
–S-S––S-S–
–S-S –
Light Chain Light Chain
Expected disulfide bonds in IgG1 Antibody Trypsin Digest
©2012 Waters Corporation 31
�8 unique S-S bonded peptides
�LC: 2 Intra, HC: 4 Intra,
�HC-HC(Hinge): 1 inter
�HC-LC:1 interCHOCHO
C L
CH 3
CH 2
– –
S-S––S-S
SS
SS
SS
SS
–S-S –
-S-S-
Humanized IgG
Light Chain (1)
Light Chain (4)
Heavy Chain (2)
Heavy Chain (3)
K K
Disulfide Containing Peptides
2:T21-3:T21
Nonreduced peptide mapping enabled ID of all canonical S-S peptides
A simple filter to only display disulfide containing peptides
©2012 Waters Corporation 32
2:T21-3:T21
2:T21-3:T21
2:T21-3:T21
MSE
Fragment Ions2:T21-3:T21
UNIFI enables researchers to focus on critical attributes of a molecule
Additional studies show there are no scrambled disulfide presence
Disulfide Bonds Report: Unifi enables researchers to focus on critical attributes of a molecule
Component Plot for S-S peptides
Analysis Information
KK
©2012 Waters Corporation 33
ComponentSummary
Analysis Workflow for mAbs
Glycan Analysis
Non-ReducedPeptide Mapping
IntactMass Analysis
Aggregate AnalysisReduced LC
Mass Analysis
ReducedPeptide Mapping
©2012 Waters Corporation 34
Peptide Mapping
Charge variantAnalysis
Mass Analysis
Reduced Deglyco HCMass Analysis
Reduced HCMass Analysis
UPLC and MS Based Approaches for Glycoprotein Characterization (Work Flows)
Intact Glycoprotein
GlycopeptidesN-Released Glycans
MW profiling(ESI MS)
PNGase F Digestion
Proteolytic Digestion
©2012 Waters Corporation 35
GlycopeptidesN-Released Glycans
Fluorescent Label
HILIC/FLR/MS
Peptide MappingUPLC-UV and UPLC-MS
Most biotherapeutic drugs are glycosylated, and the
glycan population constitutes a critical quality attribute.
Glycosylation can affect:
�Stability
�PK/PD
�Activity/Binding
�Conformation
�Immunogenic response
UPLC and MS Based Approaches for Glycoprotein Characterization (Work Flows)
Intact Glycoprotein
MW profiling(ESI MS)
PNGase F Digestion
Proteolytic Digestion
©2012 Waters Corporation 36
GlycopeptidesN-Released Glycans
Fluorescent Label
HILIC/FLR/MS
Peptide MappingUPLC-UVUPLC-MS
Peptide Mapping for:
Location of Glycosylation
O- and N-linked glycans
Glycans profiling and quantitation
Structure Elucidation
Batch-to-Batch Comparison (% Glycosylation) across the Samples
HC Peptide T26 G1FGlycopeptide Trendline Plots
% G
lycovariants
Response
% G
1F R
esponse
Peptide Mapping
©2012 Waters Corporation 37
Sample injectionsSample injections
Reduced HC Mass Analysis (G1F)
% G
lycovariants
Response
% G
1F R
esponse
% G
1F R
esponse
Sample injections
UPLC and MS Based Approaches for Glycoprotein Characterization (Work Flows)
Intact Glycoprotein
MW profiling(ESI MS)
PNGase F Digestion
Proteolytic Digestion
©2012 Waters Corporation 38
GlycopeptidesN-Released Glycans
Fluorescent Label
HILIC/FLR/MS
Peptide MappingUPLC-UVUPLC-MS
Quantitation of Individual Glycans
Profiling of glycans
Structure Elucidation
GlycoWorksTM Consumables for Released Glycan Preparation
©2012 Waters Corporation 39
(1) ACQUITY FLR ChA Ex330,Em420Range: 18464
1 2
3
4
5
6
7
8
9 1011
12
13
14 15 1617
18
19
FLR
UPLC Separation of Glycan Performance Test Standard Using BEH Glycan Column
©2012 Waters Corporation 40
Time (minutes)
10 15 20 25 30
TOF MS
4.
MS
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mmA: 100mM ammonium formate pH 4.5B: Acetonitrile75% B to 60% B over 46.5 mins, 0.5mL/min, 60°CFluorescence: λex = 330 nm, λem = 420 nm
Glycan Performance Test Standard: 2AB-labeled glycans from Human IgG mAb mixed with M5 and M6
HILIC separation is orthogonal to RP
Calibration curve
Assigning released 2-AB Glycan peaks based on GU or retention time
©2012 Waters Corporation 41
2AB-labeled Dextran Ladder Standard is used to perform retention time calibration
The Glycan Performance Test Standard is used to test the UNIFI system performance
FLR
Waters Glycan GU library Search Result
Assigning released 2-AB Glycan peaks based on GU or RT and MS
©2012 Waters Corporation 42
XIC
BPI MS
Sample list
Chromatogram, MS and summary table
UPLC/FLR
Tof MS
UINFI Glycan Report
©2012 Waters Corporation 43
RT calibration
Waters GU Library Search Results
N-Glycan Profile is Measured for Antibodysamples
� Based on GU and mass
� 24 glycan species ID
� Some batches have several sialic acid-
containing glycans (NeuAC and NeuGC) and
low level of 1,6 alpha-Galactose.
©2012 Waters Corporation 44
low level of 1,6 alpha-Galactose.
� Alpha-Gal is a potentially
immunogenic glycan.
Batch-to-Batch Comparison: some batchescontains ~1% 1,6-alpha Gal
alpha–Gal (~ 1%)
FLR InnovatorBatch
I
II
III
©2012 Waters Corporation 45
~1% of total glycan content is 1,6-alpha gal in some batches.
BatchI
XIC
mAb Screening Workflow – Core Requirements
ReductionAlkylationDigestion
Host Cell Proteins
Variant Profiling
Higher Order Structure
UPLC/ MSE
Peptide Map
Intact (mAb) Mass by LC/MS
©2012 Waters Corporation 46
Subunits PNGaseF DeglycosylationHILIC Glycan SPE2AB Label & CleanupHILIC-FLR (MS)
Stability and Formulation
Proteins
Bioanalysis
QC Testing
Released Glycan (FL/ MS)
Light Chain, Heavy Chain UPLC/ MS
总结� 由于抗体结构的复杂性与非均一性,完整高效的分析流程是抗体结构分析策略中不可或缺的重要组成部分。这种流程应当完整地包括下列组成部分:高重现性的样品制备,高效的分离手段;准确可靠并能同时定性定量的样品检测手段;以及高效准确并能快速将数据转化为资讯的科学信息工具。
� 分析流程的建立并不是其各个组成部分的简单堆砌,它应当是为了蛋白质结构分析表征而专门开发的,将各个分析步骤有机结合起来的高效实验室平台。这个实验室平台同时应该具有功能多样性,可满足蛋白质结构分析中,对不同类别不同层次的
©2012 Waters Corporation 47
平台同时应该具有功能多样性,可满足蛋白质结构分析中,对不同类别不同层次的结构表征的需求。
� 沃特世公司为蛋白质结构表征而开发的UNIFI Platform Solution, 包括完整蛋白分析流程,肽谱分析流程以及糖基分析流程等,综合了为各种分析物质而研发的专业技术,并加以整和从而形成了一系列完整流畅的分析流程。这些流程共同使用同一种仪器和软件平台,但各自拥有相对独立的数据处理软件和分离色谱柱,从而形成了相互关联但功能有别的蛋白质结构表征方案。
� UNIFI软件独有的数据库、审计追踪、报告等强大功能。
©2012 Waters Corporation 48