unifi lc ms - waters corporation...unifi system based workflow and solutions for ©2012 waters...

基于UNIFI的蛋白药物LC与MS分析表征方案

©2012 Waters Corporation 1

宋兰坤[email protected]

Biopharmaceutical Platform Solution with UNIFI 1.7

Biopharmaceutical Platform Solution

Intact Protein Mass

Peptide Mapping

DDA (Peptide & Glycan)

Xevo G2-S QTof


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


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


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


Reduced HCMass Analysis

Charge variantAnalysis

Reduced Deglyco HCMass Analysis


Aggregate Analysis

Glycan Analysis


IntactMass Analysis




Peptide Mapping


Mass 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


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


� 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


Monomer : 99.3 to 99.5%

Dimer : 0.5 to 0.7%


AggregateAnalysis

Glycan Analysis


IntactMass Analysis





AnalysisPeptide MappingMass 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


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


AB+ allows the flexibility to use either pH or salt gradients when characterizing biomolecules


pH

Ionic S

trength

(m

M)


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


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


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%


IntactMass Analysis


Aggregate Analysis

Glycan 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


Relative abundance of C-terminal lysine variants can also be compared by intact mass analysis

Batch-to-Batch Consistency%

No Lysine I II III


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


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


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


0K

IntactMass Analysis


Aggregate Analysis

Glycan Analysis







Mass Analysis


Mass analysis of Antibody heavy chain reveals the heterogeneity of the subunit


+

Reduction

LC HC

KKK

Innovator

LC

G0F +K


G0F

G0+K

Man5

G1F +K

G2F +K



IntactMass Analysis

Aggregate Analysis

Glycan Analysis





Mass Analysis



Experimental Setup for Peptide Mapping

Antibody

Denature & Alkylate

Trypsin DigestLC/MSE

Non-ReducedPeptide Map

Reduction &

Alkylation LC/MSE

Reduced Peptide Map


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.


Protein coverage was obtained

HC Protein Coverage


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


isoAsp is not a natural amino acid and can potentially be immunogenic.

Oxidation of HC Peptide T42

II


% O

xidation

Sample Injections

I


IntactMass Analysis

Aggregate Analysis

Glycan Analysis






Mass 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


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


�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


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


ComponentSummary


Glycan Analysis


IntactMass Analysis

Aggregate AnalysisReduced LC

Mass Analysis



Peptide Mapping


Mass 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



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


Intact Glycoprotein


PNGase F Digestion




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


Sample injectionsSample injections

Reduced HC Mass Analysis (G1F)

% G

lycovariants

Response

% G

1F R

esponse

% G

1F R

esponse

Sample injections


Intact Glycoprotein


PNGase F Digestion




Fluorescent Label

HILIC/FLR/MS

Peptide MappingUPLC-UVUPLC-MS

Quantitation of Individual Glycans

Profiling of glycans

Structure Elucidation

GlycoWorksTM Consumables for Released Glycan Preparation


(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


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


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


XIC

BPI MS

Sample list

Chromatogram, MS and summary table

UPLC/FLR

Tof MS

UINFI Glycan Report


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.


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


~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


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


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。

unifi lc ms - waters corporation...unifi system based workflow and solutions for ©2012 waters...

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