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Mass Spectrometry in Clinical Diagnostics Industry point of view Munich June 2012 Uwe Kobold
Mass Spectrometry in Clinical Diagnostics
Industry point of view Munich June 2012
Uwe Kobold
Where we are and where to go?
2
„Powerfull“ technology?
5) Arrived in diagnostics ?
1) Todays use in diagnostics
development ?
3) Acceptance for routine ?
Questions …
4) Regulatory aspects ?
2) Potential for routine use in
the clinical laboratory ?
3
Today in Diagnostic development
Pro
teo
mic
s
Qu
an
tita
tive
MS
Qu
ali
tati
ve
MS
Str
uc
ture
de
term
.
Sta
bil
ity i
ssu
es
Re
fere
nc
e m
eth
od
Re
fere
nc
e m
eth
od
Rare
Reag
en
t
ch
ara
cte
riza
tion
Re
fere
nc
e m
eth
od
1 2 3 4 5
Production Marketing/
Productcare
Marker
discovery
& evaluation
Reagent
development
Test
development Standardization
Mass Spectrometry inside…
4
Today MS hardware at Roche Diagnostics R&D in Penzberg
Electrospray MS
Thermo Q-exactive
Thermo Orbitrap Velos
Thermo LTQ-FT
Thermo TSQ Vantage
2 x Thermo Quantum Ultra
3 x Thermo LTQ
Thermo LCQ deca XP
Waters LC-TOF
Waters QTOF
Waters Quattro Micro
MALDI MS
ABI Voyager pro
Bruker Ultraflex TOF/TOF
EI-MS (4)
Leco Pegasus GC-TOF
PE Clarus 680 GC-MS
PE Autospec XL GC-MS
Thermo DFS double focusing sector MS
5
Tissue Cell culture Body fluid
protein
extraction
digest to
peptides
Measurement by LC-MS
Identification + Quantification of 1000´s of proteins
Hypothesis-free: open for everything / novelties
Result:
1000´s of proteins measured simultaneously, no assays required
1 2 3 4 5
Today
Discovery for novel protein biomarkers: Quantitative Proteomics
Source: Stefan Hanke 6
E:\data\...\sd_26Sept2011_a10 9/26/2011 8:56:22 PM NIST Cal 1 (21.Sept2011,SD)
Probenvorb. 21.Sept2011 6
RT: 0.0 - 21.0
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VitD3/epi-OHD3
RT: 8.8AA: 3099494
ISTD
RT: 8.8AA: 193374
VitD2
RT: 9.1AA: 402305
24,25-OH2-VitD3
RT: 6.7AA: 1455228
NL: 2.60E5
TIC F: + c APCI sid=7.00 SRM ms2 401.300 [159.200-159.400, 257.100-257.300] MS Genesis sd_26Sept2011_a10
NL: 2.66E4
TIC F: + c APCI sid=7.00 SRM ms2 407.300 [159.100-159.300, 263.100-263.300] MS Genesis sd_26Sept2011_a10
NL: 5.04E4
TIC F: + c APCI sid=7.00 SRM ms2 413.400 [159.100-159.300] MS Genesis sd_26Sept2011_a10
NL: 2.41E5
TIC F: + c APCI sid=7.00 SRM ms2 417.300 [381.200-381.400] MS Genesis sd_26Sept2011_a10
MRM trace 1
MRM trace 2
MRM trace 3
MRM trace 4
Today Easy separation of metabolites unique for HPLC-MS/MS
Determination of isobaric metabolites:
Need for chromatographic separation
Pentafluorophenyl (PFP)
Cyanopropyl (CN) stationary phase
25OH-D3 and
epi25OH-D3
on C18 RP HPLC
Today
sensitivity limits for quantitative peptide µHPLC-MS/MS
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Re
lative
Ab
un
da
nce
:
:
4.2 aMol/µl Angiotensin
On column
0 1 2 3 4 5 6 7 8 9
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0.5 fMol/µl Angiotensin
On column
0.3 fMol/µl Hepcidin in serum on column µHPLC-MS/MS
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Rela
tive A
bundance
Hepcidin
DTHFPICIFCCGCCHRSKCGMCCKT
Today examples for quantitative MS methods at Roche Diagnostics R&D in Penzberg
Methods available
HbA1c
25OH Vitamin D3
25OH Vitamin D2
Testosterone high sensitive
Progesteron
Gentamycine
Vancomycine
Drug of abuse testing
Benzoylecgonine
Propoxyphen
Imipramine
Phencyclidine
Benzodiazepines
Methadone
Methaqualone
THC
Amphetamines
Opiates
Barbiturates
Benzodiazepines – Screening
Research methods and methods in development
Hepcidin
Vitamins in culture media
High sensitive protein MS
Folates
Drugs in oral fluids
24,25(OH)2 Vitamin D3
Epi 25OH Vitamin D3
Immunosuppressents
Digoxin/Digitoxin
etc.
1 2 3 4 5 9
Today request for more sensitivity
6-40 nmol/L
25-150 nmol/L
10-9 M
60 fmol/10µL 2-20 ng/mL Folate
250 fmol/10µL 10-60 ng/mL Vitamin D
LOQ
Amount in10µL sample
Concentration Analyte
< 2 amol/10µL < 0.2 pmol/L <2-125 pg/mL NT-proBNP
(MW~9kDa)
< 4 pmol/L
10-12 M
< 40 amol/10µL < 0.1-100 ng/mL Troponin I (MW~23kDa)
New diagnostic markers are in many cases below <10-12 M
10 1 2 3 4 5
Potential Trend in patent applications for MS & Clinical Diagnostics
1 2 3 4 5 11
Potential requirements routine analyzer serum work area
Samples
Reagents
Results
easy – fast – reliable
fully regulatory compliant
1 2 3 4 5 12
Potential Requirements
Ready-to-use packs Information
Sample
Sample preparation
module
Measuring module
• HPLC unit
• MS unit
Data module
Analysis data Sample ID data
fully regulatory compliant 1 2 3 4 5
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Potential hypothetical example HbA1c
potential reagents, hardware, software
Hardware/Software
Liquid Handling
HPLC Pump
Injector
Mass Spectrometer
Software
Consumables
Enzyme
Internal Standards
Calibrators
Controls
Solvents
Column
1 2 3 4 5 14
Potential example HbA1c Runtime and precision HPLC-MS and MS/MS
CVs < 2%
Clinical Chemistry 46: 281-283, 2000
4.0 5.0 6.0 7.0 8.0 9.0 0
100 6.90
HbA1c
Hexapeptide ,
m/z 429
4.0 5.0 6.0 7.0 8.0 9.0
Time (min)
0
100
Rela
tive
Ab
un
da
nc
e
5.63
HbAo
Hexapeptide ,
m/z 348
IFCC reference method
Clinical Chemistry 34:1944-1951, 1997
CVs < 2%
8 minutes 3 minutes = 20 samples/h
1 2 3 4 5 15
Potential Comparison with established routine assays: example HbA1c
• Cobas Integra 800
300 samples/hour
• Roche Hitachi 917
400 samples/hour
1 2 3 4 5 16
Potential
example HbA1c
Throughput:
300 samples/h (12 sec/sample)
Robustnes
Certifation: MPG, FDA, …
Precission:
CVs < 2%
1 2 3 4 5 17
Acceptance for routine? Experts needed – backup need
“You need an expert who is familiar with the technology ~
to be in the background in case of problems”
“If a vacuum pump fails, by the time an engineer comes out,
orders parts, and comes back, altogether it can take 10 days.
You have to have a plan for ongoing operations.”
Quelle:
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Acceptance for routine?
the fear factor
“There‟s a fear factor of „how am I going to work with this non-FDA-approved
technology? How do I work with a lab-developed test?‟ That‟s intimidating for many
non-academic labs”
Quelle:
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Regulatory aspects
Accountabilities according to Medizinproduktegesetz
• Manufacturer is accountable for his product and the obtained results according to the German Medizinproduktegesetz (MPG; medical device law) if the costumer follows the intended use.
• The original manufacturer is not accountable, if the product is used not for the inted use. Then the responsibilty is on the user side!!
20 1 2 3 4 5
Regulatory aspects Who is accountable?
Example 1
• Different Manufacturer
– Hardware Company A
– Reagents Company B
• Both products are independently CE marked
• Shared Accountability according to MPG
• However
• User becomes a manufacturer of an Diagnostic Device (Reagent plus Hardware)
• User is responsible for the whole system
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Regulatory aspects Take over of accountability
Example 2
• Full service from one manufacturer
– Complete system: Application, Reagent, HPLC, MS, Software
• Harmonized modules with CE mark
– Manufacturer is responsible for the whole system according to MPG
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Arrived in clinical diagnostics? Performance of todays MS
Technology viewpoint Diagnostic needs viewpoint
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Technology viewpoint
Dramatic improvements during the last years
• sensitive
• robust
• convenient
• universal
• cheap
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Diagnostic needs viewpoint
We need…
1. Higher sensitivity
2. More robustness
3. Higher convenience
4. Faster turn around times
5. Full regulatory compliance
YES …… BUT
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Arrived in clinical diagnostics? Strength and weakness
Strength
Specific
Multiplexing
Flexible
Simple development
Cheap reagents
Weakness
Maintenance intervals (costs)
Regulatory hurdles
Well trained staff required
Throughput
Protein in low concentration
1 2 3 4 5
Powerfull technology?
26
Arrived in clinical diagnostics?
MS for diagnostics development
MS for clinical routine in the core lab
MS for special applications
Mass Spectrometry inside…
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MS found its place in the product family,
for broad routine use several hurdles have to be overcome
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Innovation für die Gesundheit
