The Identification and Quantification
of Residual Host Cell Proteins
(HCPs)
Steve Taylor
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Overview
ƒ In purified
product there
is a high
concentration
of ‘product’
‘p od ct’
proteins low
concentration
of ‘host’
proteins
ƒ Waters can
Identify and
Quantify these
with UPLC-MSE
©2009 Waters Corporation | COMPANY CONFIDENTIAL
2
HCP
Background to HCPs and the
Guidelines of the EU Regulatory
Authority
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Host Cell Proteins (
(HCPs)
)
ƒ Recombinant Proteins produced in host
cells
ƒ Proteins from cells can co-purify with
therapeutic protein of interest
—e.g. Chinese
Chi
Hamster
H
t Ovary
O
(CHO) cell
ll
proteins in recombinant monoclonal
antibody therapeutics
ƒ Purification steps should remove
contaminants. Low levels can remain
because of
—Poor process control
Process changes: can affect HCP
—Process
pattern and abundances
Biopharm International, Volume 13, Number 6, pp. 38-45, May 2008
©2009 Waters Corporation | COMPANY CONFIDENTIAL
4
Guidelines Governing
g HCPs
ƒ Safety drives the need for removal/minimization
—Link
Link between HCPs and immunogenicity
ƒ European regulations in effect since 2007
—‘6
6.2
2 Validation of the purification
procedure - …. The ability of the purification
process to remove other specific
p
p
contaminants
such as host-cell proteins … should also be
demonstrated’
—ICH Guidelines: 2009 review in progress
(http://www.emea.europa.eu/pdfs/human/bwp/BWPworkprogramme.pdf)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
5
Importance
p
of HCPs – approval
pp
ƒ 2008: approval of follow-on
biologic/ biosimilar (EU; USA)
Diagramatic
representation of amino
acid sequence of human
growth hormone
— Initially the application was turned
down due to a potential
immunogenetic effect due to HCPs
— Issue was resolved before drug
release – positive opinion given
— ‘The cause of immunogenicity
was linked to excess host cell
protein contamination, which
was resolved by the
manufacturer
f t
with
ith additional
dditi
l
purification steps’.
(http://www.pubmedcentral.nih.gov/articlerender.fcgi?
artid=2638545)
artid
2638545)
Image source: http://www.rxlist.com/omnitrope-drug.htm
©2009 Waters Corporation | COMPANY CONFIDENTIAL
6
Importance
p
of HCPs – failure
ƒ HCPs and approval problems for some Biosimilars:
— Application
pp cat o by biosimilar
b os
a company
co pa y for
o Interferon
te e o Alfa
a 2a
a (HepC)
( epC)
— Marketing permission rejected 2006:
— “The reasons for the rejection by the EMEA included quality
and clinical differences between [the biosimilar product] and
the reference product, … inadequate validation of the
process for the finished process and insufficient
validation of immunogenicity testing.”
testing ”
Liver Damage
from Hepatitis
Image Source : Copyright © 1996, 1997 University of Pittsburgh http://tpis.upmc.edu/tpis/HB/H00030m.html
©2009 Waters Corporation | COMPANY CONFIDENTIAL
7
Challenges
g
of HCP Analysis
y
ƒ Thousands of possible protein contaminants
ƒ HCPs can be present at extremely low levels
o Typically ppt to ppm (relative to biotherapeutic)
o Guidelines suggest monitoring to ppm (1-100ppm)
ƒ Developing methods is expensive and time consuming
ƒ Business Impacts of Failure to identify and remove
contaminants:
o Can reduce drug efficacy
o May lead to adverse events
o Drug development and introduction delays
o Longer cycle to introduce process improvements
o Perceived product quality issue = competitive disadvantage
o Kill a promising candidate
©2009 Waters Corporation | COMPANY CONFIDENTIAL
8
Main Goals of Waters Host Cell
Protein Analysis
y
Develop a method
— To identify,
— Quantify and
— Monitor HCPs from recombinant means
ƒ Results must have a means of validating the results (e.g.
peptide sequence; concentration confirmation)
ƒ Needs to be complementary/ compatible with existing
methods
ƒ Needs to provide improvements compared to existing
methods (generality; efficiency; speed)
ƒ Needs to provide cost-effective benefits for process
improvements for Waters customers
©2009 Waters Corporation | COMPANY CONFIDENTIAL
9
HCP
Current Methods for Host Cell
Protein Analysis
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Comparison
p
of current HCP Methods
Narrow dynamic range (<100)
Biopharm International, Volume 13, Number 6, pp. 38-45, May 2008
©2009 Waters Corporation | COMPANY CONFIDENTIAL
11
EFFICIENCY
ƒSimultaneous i.d. and Quan
of proteins
ƒEfficient assay and use of
resources
SPEED
ƒSignificantly shorter
development time for assay
ƒAbility to provide high
throughput/ flexible monitoring
assay
Why
h UPLC/MS
C/ SE off HCPs?
C ?
ACCURACY
ƒAccurate quan of HCPs in
complex mixture
ƒQuan over > 4 orders of
magnitude
©2009 Waters Corporation | COMPANY CONFIDENTIAL
GENERALLY APPLICABLE
ƒHCPs do not need to be known
prior to analysis
ƒCan be widely applied and easily
modified
Attributes Needed for Analysis of
HCPs – User Expertise
p
User Expertise Needed for Routine Use
LC/MS Tof
High
LC/UV
Low
ELISA:
Very Low
Gel and Blot:
Low
LC/MS Quad
Reasonably Low
©2009 Waters Corporation | COMPANY CONFIDENTIAL
13
Attributes Needed for Analysis of
HCPs - Quantitation
Q
Ability to Quantify over wide dynamic range
ELISA:
Poor
LC/UV
2 - 3 orders
Good
LC/MS
4 orders
Good
Gel and
Bl t
Blot:
Poor
©2009 Waters Corporation | COMPANY CONFIDENTIAL
14
Attributes Needed for Analysis of
HCPs - Sensitivity
y
Ability to Detect at very low levels
LC/UV:
Acceptable
(high ppm)
LC/MS
Good (ppm)
ELISA:
Excellent
(ppt)
Gel and
Blot:
Variable
©2009 Waters Corporation | COMPANY CONFIDENTIAL
15
Attributes Needed for Analysis of
HCPs - Interpretation
p
User Interpretation required for Analysis
LC/UV
Objective by
RT
ELISA:
Subjective
LC/MS
Highly
Objective
Gel and
Blot:
Subjective
©2009 Waters Corporation | COMPANY CONFIDENTIAL
16
Attributes Needed for Analysis of
HCPs - Certainty
y
Unambiguous Identification of HCPs
LC/UV:
High with
SOP
ELISA:
Low
LC/MS
Extremely
High
Gel and Blot:
Acceptable
©2009 Waters Corporation | COMPANY CONFIDENTIAL
17
Attributes Needed for Analysis of
HCPs - Certainty
y
Method Flexibility
LC/UV:
Flexible
ELISA:
Very
Inflexible
LC/MS
Extremely
Flexible
Gel and Blot:
Flexible
©2009 Waters Corporation | COMPANY CONFIDENTIAL
18
HCP
Summary of Waters Host Cell
Protein Methodology
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Comparison
p
to Proteomics
Similarity to proteomics applications
- Similar tools can be used with minor changes
- Complex Samples by tryptic digest
g and rules for identifying
y g
- Same data mining
- Databases used
- MSE acquisition
Differences:
- Greater need for dynamic range (>4)
- Need
d to cope with
h high
h h product
d
concentration and
da
small amount of HCP (ppm)
- Not normally sample limited
- Databases can be tailored because Host is known
©2009 Waters Corporation | COMPANY CONFIDENTIAL
20
Tools Available for HCP Analysis
y
Informatics
ƒ PLGS and IdentityE: validated protein identification
reducing
d i
ffalse-positive
l
iti
space.
ƒ BiopharmaLynx 1.2 for automated sequence coverage
and confirmation of primary structure of biomolecules
(intact mass; peptide mapping)
ƒ VerifyE for the determination of the most appropriate
peptides for quantification (by MRM)
Instrumentation
ƒ NanoUPLC with 2D RP-RP – more reproducible
chromatography (greater sensitivity)
ƒ Synapt/ XevoQtof – accurate mass MSMS
ƒ TQD/ Xevo TQ – high dynamic range quantitation
Chemistry
ƒ Rapigest: aids tryptic digestion
ƒ PST/ BEH: Peptide Separation Columns
ƒ HILIC (‚normal phase‘); Glycan columns
©2009 Waters Corporation | COMPANY CONFIDENTIAL
21
General Methodologies
g
IdentityE to discover proteins
- Peptide sequences matched (dB)
- Confidence ranking of identification
BiopharmaLynx to
- Compare samples vs control
- Monitor/
M it / quan modifications
difi ti
-
ExpressionE to quantify proteins
- Measure of amount
- Established
E t bli h d protocol
t
l
- Generally applicable
-
(e.g. glycoforms)
Confirm peptide sequence with MSE
VerifyE for ‘signature’
signature peptides
- Relevant Peptides obtained for MRMs
- Output of MRM method for Tandem Quad
©2009 Waters Corporation | COMPANY CONFIDENTIAL
22
HCP
The Application of 2D
nanoAcquity Chromatography
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Optimisation
p
of 2D Chromatography
g p y
FIRST DIMENSION:
ƒ 1 mm x 0.5 cm X
X-Bridge
Bridge packed with BEH130, 5
µm; 10 µL/min at pH 10 to elute all peptides.
— HIGH RESISTANCE to extreme pH
ƒ Trap column
col mn fo
for this research
esea ch project
p oject 500 µm
mx
2 cm packed with Symmetry C18, 5 µm
— High loading capacity
SECOND DIMENSION:
ƒ 300 µm x 15 cm with BEH130
BEH130, 1.7
1 7 µm; 4
µL/min
ƒ Standard ESI probe with narrow bore capillary
©2009 Waters Corporation | COMPANY CONFIDENTIAL
24
MSE Alternating High/Low
Energy
gy Acquisition
q
MS
Precursor
MSE
Fragments
Retention Time
©2009 Waters Corporation | COMPANY CONFIDENTIAL
25
Yeast
Enolase
MS
TIME
MSE
©2009 Waters Corporation | COMPANY CONFIDENTIAL
26
15 Seconds
Chromatographic Window
Product Mass
Prec
cursor Mass
Time-Aligned
TimePrecursor/Product
/
Ion list
retention time
MH+
Ret.Time
Volume
ChargeStat
e
1189.5802
46.71
78430
1.98
765.3742
46.67
449
1
522.2606
46.67
554
1
800.4481
46.67
3754
1
963.5187
46.69
3658
1
515.3250
46.70
2325
1
687.3742
46.70
2351
1
1100.5773
46.71
1112
1
822.4154
46.71
436
1
781.4823
46.71
163
1
896 5183
896.5183
46 72
46.72
675
1
685.3210
46.72
862
1
1009.6112
46.74
125
1
498.3296
46.75
709
1
432.2357
46.75
356
1
906.5477
46.76
364
1
Precursor/Product Ion List
©2009 Waters Corporation | COMPANY CONFIDENTIAL
27
Waters IdentityE High
Definition Proteomics System
y
‘C il
‘Capilary’
’ scale
l Chromatography
Ch
t
h
nanoAcquity UPLC system
Mass Spectrometry
SynaptTM HDMSTM
with 2D Technology
Informatics
IdentityE Software
ProteinLynx Global Server
©2009 Waters Corporation | COMPANY CONFIDENTIAL
28
Comprehensive Peptide Ion
Accounting
g
1
2
3
4
5
©2009 Waters Corporation | COMPANY CONFIDENTIAL
29
HCP
Methodology
h d l
ffor Host Cell
ll Protein
i
Monitoring by Tandem Quadrupole
Once HCPs have been established
a high
high-throughput
throughput method can be
developed for process monitoring.
©2009 Waters Corporation | COMPANY CONFIDENTIAL
VerifyE
y flow diagram
g
2 3 .8 0
4 1 8 .7
100
2 2 .4 5
4 5 8 .7
Discovery phase
LC-MSE data
Acquisition as above
3 3 .1 3
4 8 4 .7
2 7 .3 7
4 2 2 .2
2 6 .9 9
5 4 0 .2
2 4 .9 3
4 5 9 .7
VerifyE data processing
(N) proteotypic pep
(X) trans per peptide
2 9 .1 2 2 9 .9 3
6 2 6 .3 4 0 7 .7 3 0 .7 3
5 8 2 .3
1 9 .4 0
4 0 6 .2
%
3 3 .2 4
7 2 4 .3
2 4 .5 4
4 6 1 .7
2 0 .8 7
4 1 1 .7
1 8 .7 4
5 5 1 .2
2 6 .7 7
4 1 6 .7
2 6 .2 6
4 3 5 .2
2 1 .6 0
7 2 2 .2
2 6 .1 6
5 3 1 .7
2 2 .8 6
4 4 7 .2
2 5 .8 9
5 5 9 .2
3 1 .3 5
5 2 7 .2
2 8 .2 2
4 0 7 .7
2 8 .2 9
5 3 6 .7
3 1 .9 0
7 2 1 .8
3 3 .4 4
5 9 8 .3
1 6 .9 3
4 8 8 .5
1 7 .4 7
5 7 5 .2
2 0 .2 2
3 7 3 .2
3 2 .2 8
6 6 9 .7
3 0 .3 5
6 2 5 .3
1 7 .9 6
4 1 9 .9
3 5 .8 4
7 9 0 .9
3 3 .7 6
7 4 0 .3
3 6 .9
90
7 0 0 .3
3 5 .7 0
7 8 9 .8
3 9 .1 1
4 9 9 .2
4 1 .7 9
7 8 2 .3
0
T im e
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
2 6 .0 0
2 8 .0 0
3 0 .0 0
3 2 .0 0
3 4 .0 0
3 6 .0 0
3 8 .0 0
4 0 .0 0
4 2 .0 0
Automatically generated
Xevo TQ MRM exp file
Verification- MRM transition monitoring
©2009 Waters Corporation | COMPANY CONFIDENTIAL
31
MRM development for Targeted
Monitoring
g
ƒ Use MSE data to develop MRM
assay
ƒ Use in high-throughput
monitoring and absolute
quantification of HCPs
Xevo TQMSTM
MS1
Static
ƒ Xevo TQ with selected MRMs
Collision Cell
MS2
Static
©2009 Waters Corporation | COMPANY CONFIDENTIAL
32
Applying Proteomics Workflows
to HCP Analysis
y
ƒ Workflow Overview:
—Shotgun enzymatic digestion of sample into
peptides
—1D
1D or 2D LC/MSE with IdentityE to DISCOVER
contaminant proteins
—2D
2D for more loading capacity
—Develop proprietary host cell protein database
—(Hi3
(Hi3 for absolute quantitation, label
label-free)
free)
—Data mined for MRMs
Quad for absolute
—Transfer to Tandem Q
quantitation (e.g. labeled peptides)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
33
2D Chromatography
g p y Factors
ƒ 2D Chromatography requires optimisation because:
Column loading is non
non-linear
linear (more loading
—Column
does not equate to more dynamic range)
—Product is present in much higher concentrations
- 2D app
approach
oa h means low-level
lo le el imp
impurities
ities
Gilar M. et. al, J.
Sep. Sci. 2005,
quantifiable despite disparity in
28, 1694-1703
concentrations
ƒ Chemistries specifically selected for RP/RP 2D
approach:
—Retention
Retention time models applied (based on
hydrophobicity scale of tryptic peptides)
—All Chemistries readily available (but dimensions
adapted)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
34
Host Cell Protein Analysis of
Biopharmaceutical
p
Product
ƒ 1D Chromatography (75 um scale)
—0
0.05
05 ug of
o product
p oduct digest
d gest loaded
oaded for
o peptide
pept de mapping
app g
— 90 min gradient (5-40% acetonitrile)
ƒ 2D RP-RP (High/Low pH) Chromatography
Gilar M. et. al, J.
Sep. Sci. 2005,
28, 1694-1703
— 5 ug (80 pmol)
l) off product
d t digest
di
t (over)loaded
(
)l d d + 100 fmol
f
l ADH
+ 1 fmol BSA
— 1st Dimension (pH 10): 5 or 10 step gradient (0 - 45% acetonitrile)
— 2nd Dimension (pH 2.6): 90 min gradient (5 - 40% acetonitrile)
UPLC: nanoACQUITY® 2D UPLC®
QTof: SYNAPT MS (MSE mode)
D t
Data:
PLGS 2
2.4
4 (Identity
(Id tit E)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
35
Diagram
g
of 2D setup
p at higher
g
scale
Online dilution of 1D flow
to Trap column – change
pH a
p
and
d therefore
t e e o e selectivity
se ect ty
1D pH=10. flow 10 µL/min. XBridge high
resistance to pH regime. Mobile phase
20 mM ammonium formate in water
(Solvent A) and ACN (Solvent B). Five
Fractions (to 50.0% B).
2D pH=2.4. 0.3 mm x 150 mm BEH
C18 1.7 µm, Flow at 4 µL/min. 90
min gradient from 3 to 40%
acetonitrile
t it il (0
(0.1%
1% FA-formic
FA f
i acid).
id)
Trap: 5-µm
Symmetry
C18 trap peptides
washed on to
2D column.
©2009 Waters Corporation | COMPANY CONFIDENTIAL
36
2D HPLC using High/Low pH RPLC
pH 10.0
20 mM ammonium formate
0-42%
0
42% acetonitrile in 5 or 10 steps of 15 min
TIC
4.37e8
100
pH 10
18 95
18.95
41.26
25.68
%
35.85
14.03
13.21
10.38
5.70 6.77
4.10 5.21
16.41
11.73
9.64
37.65
29.41
15.79
8.53
25.92
22.39
18.58
18 21
18.21
41.92
29.00
39.58
35.48
21.04
19.89
24.20
0-56%
1 B in 70 minutes 20 mM NH4OH pH 10
2.50
5.00
7.50
10.00
Bovine_Hemoglobin_Digest_Stored_091803_1
12.50
15.00
17.50
neutral
100
20.00
22.50
25.00
acidic
30.00
32.50
35.00
37.50
28.55
acidic
basic
pH 2.6
27.50
40.00
Time
42.50 1: Scan
45.00ES+
TIC
4.51e9
basic
18 75
18.75
23.86
27.00
26.68
%
17.36
26.51
8.91
16.30
10.99
22.79
13.24
4.70
31.41
pH 2.6
0.2% Formic acid
0-42% acetonitrile in 90 min
22.39
11.40
4.29
35.05
30.68
6.29
11.93
19.61
19.93
14.09
26.06
34.27
36.19
1
Gilar M. et. al, J. Sep. Sci. 2005, 28, 1694-1703
©2009 Waters Corporation | COMPANY CONFIDENTIAL
37
2D Chromatograms
10 Fractions,, 5 μg Loaded
One peptide may
appear in multiple
Ch
Chromatographic
hi
steps - ‘merge’ step
to create coherent
fractions
45% ACN
23.6% ACN
20 8% ACN
20.8%
18.9% ACN
17.4% ACN
15.9% ACN
14.5% ACN
13.0% ACN
11.7% ACN
8.2% ACN
©2009 Waters Corporation | COMPANY CONFIDENTIAL
38
HCP
Example Results from
Biosimilar of Trastuzumab
©2009 Waters Corporation | COMPANY CONFIDENTIAL
HCPs from Biosimilar of Trastuzumab
(Non
(
European
p
Production)
)
Methodology for Greater Confidence:
ƒ R
Random
d
peptide
id sequences added
dd d as Decoy
D
strategy to
ensure identified peptides real = Total 27,216 entries in
database created
— 13,600 entries from Swissprot for Golden Hamster and Mouse
(homologs)
— 6 protein sequences from spiked in proteins: LA, ADH, PHO, BSA,
ENL, porcine trypsin,
— 2 sequences from TrastuzumAb (heavy and light chains)
q
number of random sequences
q
as known entries ((13,608))
— Equal
ƒ False Positive Rate of Protein Return: 5% (user adjustable)
ƒ Concentration range found here was 10 to 50 ppm relative to
therapeutic
ƒ Lower confidence hits (nearing random) not reported
©2009 Waters Corporation | COMPANY CONFIDENTIAL
40
HCPs from Biosimilar of Trastuzumab
(Non
(
European
p
Production)
)
ƒ Host cell: Chinese Hamster Ovary (CHO)
ƒ Database with combination of all the mouse and
hamster protein sequences listed in the SwissProt database (http://www.expasy.ch/sprot/)
—Chinese Hamster database held privately so
homology database used (Golden hamster;
M
Mouse)
)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
41
HCPs from Biosimilar of Trastuzumab –
confident assignments,
g
, 1010-50ppm
pp range
g
©2009 Waters Corporation | COMPANY CONFIDENTIAL
42
PLGS Project
j
view of proteins
p
id’d
ƒ Listing of Proteins
— Accession details
— Names and
sequences
generated
— Confidence ranking
ƒ Spectral overview
(can be zoomed)
ƒ Sequence information
for identified protein
©2009 Waters Corporation | COMPANY CONFIDENTIAL
43
Comprehensive
p
Info from PLGS
Sequence Information available even on low level proteins
©2009 Waters Corporation | COMPANY CONFIDENTIAL
44
VERIFYE
…Data WorkWork-Flow
IDENTITYE (Discovery
Data) Input .csv/.txt
Proteotypic Peptide Filters Efficient
Transition Filters
Retention Time
Optimization
Scouting Run
UPLC/MRM
UPLC/MRM (& TargetLynx) Method
File Creation
Targeted (MRM)
Quanpedia dB
Targeted (DDA) .exp
File
OPTIMISED TARGET PROTEIN
ANALYSIS UPLC/MRM
©2009 Waters Corporation | COMPANY CONFIDENTIAL
45
VERIFYE
…Proteotypic Peptide Review
…Generation of MRM Methods
©2009 Waters Corporation | COMPANY CONFIDENTIAL
46
Verify
yE to find appropriate
pp p
p
peptides
p
©2009 Waters Corporation | COMPANY CONFIDENTIAL
47
Reproducibility of the MRM assay:
MIX-5 p
protein digest
g
ƒ RSD = 3%
ƒ 1 picomole BSA digest on
column
ƒ Reproducible Chromatography
ƒ Reproducible
R
d ibl RT
RTs
©2009 Waters Corporation | COMPANY CONFIDENTIAL
48
Reproducibility of MRM assay: MIX-5 proteins
spiked
p
in Trastuzumab and digested
g
ƒ RSD = 3%
ƒ 1 picomole BSA digest on
column
ƒ Reproducible
R
d ibl Ch
Chromatography
t
h
ƒ Reproducible RTs
©2009 Waters Corporation | COMPANY CONFIDENTIAL
49
Reproducibility of the MRM assay:
MIX-5 protein digest
ƒRSD = 8%
ƒ200 fmoles ENL digest on column
ƒReproducible Chromatography
ƒReproducible RTs
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Reproducibility of MRM assay:
MIX-5 proteins spiked in Trastuzumab and
digested
g
ƒ RSD = 13%
ƒ 200 fmoles ENL digest on column
ƒ Reproducible Chromatography
ƒ Reproducible
R
d ibl RT
RTs
©2009 Waters Corporation | COMPANY CONFIDENTIAL
51
Summary of Workflow advantages
for HCP Analysis
y
ƒ Workflow Models:
— UPLC-MSE is well-established
— Proteomics tools already exist and are developing (e.g. HDMSE)
ƒ Applications Benefits:
— Confident Identification of individual HCPs – with ranking of
confidence
— Quantitation of each identified HCP
o Label-free
Label free with discovery stage (Synapt/ XevoQT)
o Using isotopically labelled peptides (XevoTQ/ TQD)
— Much faster development time than immunoassay
— Provide a multi-purpose platform for many other tasks
— Sensitivity levels comparable to ELISA (low ppm)
— Also applicable to subunit (recombinant) Vaccines
©2009 Waters Corporation | COMPANY CONFIDENTIAL
52
Conclusions
ƒ The 2D-LC/MSE setup is able to identify low abundance
protein contaminants p
p
present in biopharmaceuticals
p
over
more than 4 order of magnitude
ƒ The 2D-LC
2D LC setup using the second chromatographic
dimension provides the sensitivity and robustness required
for HCP analysis
ƒ
A high-throughput MRM assay on the Xevo TQ MS can
quantify
q
y these protein
p
impurities
p
(absolute
(
quantification
q
can be done using isotopically labeled peptides)
ƒ The combination of 2D-LC/MS
2D LC/MSE and Xevo TQ MS provides a
total system solution for HCP analysis
©2009 Waters Corporation | COMPANY CONFIDENTIAL
53
Acknowledgments
g
ƒ Waters Biopharmaceutical Development:
ƒ Catalin Doneanu
ƒ Hongwei Xie
ƒ Keith Fadgen
ƒ Martha Stapels
ƒ Jim Kehoe
ƒ Weibin Chen
ƒ Scott Berger
©2009 Waters Corporation | COMPANY CONFIDENTIAL
54