Reliable Amino Acid Analysis using the AdvanceBio AAA · PDF fileReliable Amino Acid Analysis...
Transcript of Reliable Amino Acid Analysis using the AdvanceBio AAA · PDF fileReliable Amino Acid Analysis...
M. Sundaram Palaniswamy, PhD
Application Scientist
4/19/2017
Agilent Confidential
1
Reliable Amino Acid Analysis using the AdvanceBio AAA kit
and automated derivatization
4/19/2017
Agilent Confidential
2
Agenda
1. AdvanceBio Amino Acid Solution
2. Performance data
3. Applications (Biopharma)
4. Summary
5. Q&A
Some Basics
• Amino Acids are the building blocks of proteins
• Amino Acids require derivatization to be detected by UV or FL
- OPA/FMOC, Ninhydrin, Dansyl chloride, PITC
• Derivatization can be done pre-column or post column
- OPA/FMOC and Dansyl chloride, PITC are common reagents used for pre-column
- Ninhydrin is common for post column methods
• Analysis of AA can be done by several methods:
- GC, CE, HPAE-PAD
- LC/UV/FL, LC/MS
4/19/2017
Agilent Confidential
3
Why is Amino acid analysis important?
Amino Acid Analysis is
• Important for protein and peptide identification and quantitation
• Part of reverse-phase characterization in biopharma.
• Required by the FDA
• Important for monitoring cell culture media
• Used for the analysis of metabolic Intermediates
4/19/2017
Agilent Confidential
4
The Agilent Amino Acid Analysis solution
Ready to use
AdvanceBio AAA kit
(Standards and
Reagents) All Agilent LC
systems including
Infinity II systems
AdvanceBio AAA
Columns
Fast and rugged
2.7µm
0.5µm
1.7µm
4/19/2017
Agilent Confidential
5
Online derivatization with OPA and FMOC - Fully automated in the Autosampler
Ortho-phthalaldehyde (OPA) for primary AA
9-fluorenylmethyl chloroformate (FMOC) for secondary AA
- Increase precision
- Eliminates manual
process
Automated
in ALS
Derivatization for:
1. Separation of polar AA on RP-phase
2. Detection by UV and FL
4/19/2017
Agilent Confidential
6
4/19/2017
Agilent Confidential
7
Principles of Derivatization
Post Column Derivatization:
Derivatization Pump Autosampler Detector
Cation exchange
Ninhydrin or
Fluorescamine or
OPA
The historic Gold Standard of dedicated Amino Acid Analyzers
UV/Vis
FLD
Pre Column Derivatization:
Pump Autosampler
Derivatization
Detector
Reversed Phase
a) Online in ALS
- OPA, FMOC or OPA/FMOC
b) Offline
- PITC
- Dansyl-Cl
- Benzylisothiocyante etc.
UV
FLD
Major Column Derivatization techniques
AdvanceBio AAA solution – Adding Value
• AAA part of the BioLC workflow
• Proven LC/UV solution
• programmable auto sampler, reducing complexity
• pre-column derivatization
• Column Based on Poroshell HpH Chemistry
• resolution & lifetime at high temperature and pH
• Columns batch tested with AA standards
• FMOC/OPA reagents in a kit under one part number for ease of
ordering.
4/19/2017
Agilent Confidential
8
The Product: AdvanceBio Amino Acid Analysis Solution
• Two column dimensions (3.0x100, 4.6x100) and associated guard columns
• 1 part number for all the required reagents (OPA/FMOC and standards) for
pre-column derivatization as a kit; AdvanceBio AAA Reagents kit
• Established methods using Agilent Analytical LC Systems
• Detailed product flyer, how to guide, instructions for use and application
notes for Bio-applications
4/19/2017
Agilent Confidential
9
Official Launch: Mar 1, 2017
Column Part Numbers and Configurations
ID (mm) Length (mm) Ref. Product PN
3.0 100 AdvanceBio AAA 695975-322
4.6 100 AdvanceBio AAA 655950-802
3.0 50 AdvanceBio AAA
guard (3pk)
823750-946
4.6 50 AdvanceBio AAA
guard (3pk)
820750-931
4/19/2017
Agilent Confidential
10
Components of the AdvanceBio AAA Reagent Kit
Material p/n Description
5190-9426 Kit
5061-3339 Buffer, Borate 100ml/BT
5061-3337 FMOC reagent 10 ampoules 1ml ea for
AAA
5061-3335 OPA reagent, 10 mg/ml, 6 ampoules
5062-2479 Dithiodiproprionic
5061-3330 AA, standard 1nmol 10/PK
5061-3331 AA, standard 250PMOL 10/PK
5061-3332 AA, standard 100PMOL 10/PK
5061-3333 AA, standard 25pmol 10/PK
5061-3334 AA, standard 10pmol 10/PK
5062-2478 AA supplements, 1g each
Kitted in worldwide LCs as
made to order under
AdvanceBio branding
4/19/2017
Agilent Confidential
11
Chromatographic parameters Chromatographic Parameters and materials.
1. Flow rate – 1.5 mL/min for 4.6 mm and 0.62mL/min for 3 mm ID respectively
2. Injection volume – 1µl with needle wash at the port for 7s
3. Column temperature - 40ºC
4. Detection wavelength – UV-Vis (338 and 262nm) and FLD (Ex-340nm and Em- 450nm)
5. Samples- Agilent AAA standards, media samples and protein hydrolysate standards
Gradient programme.
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 end
4/19/2017
Agilent Confidential
12
4/19/2017
Agilent Confidential
13
Fast and rugged Amino Acids Separation
• The AdvanceBio AAA columns provides the speed and resolution like a sub-2 μm
column with up to 50% less backpressure
• More forgiving for dirty samples, due to 2 μm frits
• Unique-chemically modified for high pH stability and column life time
• Guard column options that reduce your operating costs by extending the life of
columns
Unique-chemically modified
for high pH stability
Names and Order of Elution for OPA and FMOC derivatives of AA
Primary AA
Secondary AA
4/19/2017
Agilent Confidential
14
Peak # AA Name AA Abbreviation Derivative Type 1 Asparic Acid ASP OPA
2 Glutamic Acid GLU OPA
3 Aspraragine ASN OPA
4 Serine SER OPA
5 Glutamine GLN OPA
6 Histidine HIS OPA
7 Glycine GLY OPA
8 Threonine THR OPA
9 Arginine ARG OPA
10 Alanine ALA OPA
11 Tyrosine TYR OPA
12 Cysteine CYS-CYS OPA
13 Valine VAL OPA
14 Methionine MET OPA
15 Norvaline (IS) NVA OPA
16 Tryptophan TRP OPA
17 Phenylalanine PHE OPA
18 Isoleucine ILE OPA
19 Leucine LEU OPA
20 Lysine LYS OPA
21 Hydroxyproline HYP FMOC
22 Sacrosine (IS) SAR FMOC
23 Proline PRO FMOC
Retention time and area RSD precision for AA_ 4.6 x 100 mm column_100pmol (six replicates)
Amino Acids RT RSD
(%)
Area RSD (%)
1. Aspartic acid 1.270 1.066
2. Glutamic acid 0.973 1.85
3. Asparagine 0.605 1.79
4. Serine 0.629 1.82
5. Glutamine 0.470 1.56
6. Histidine 0.430 1.22
7. Glycine 0.477 1.92
8. Threonine 0.440 1.95
9. Arginine 0.251 2.15
10. Alanine 0.280 3.06
11. Tyrosine 0.128 1.65
Amino Acids RT RSD
(%)
Area RSD (%)
12. Cystine 0.067 1.9
13. Valine 0.084 2.47
14. Methionine 0.073 1.82
15. Norvaline 0.073 1.72
16. Tryptophan 0.054 1.57
17. Phenylalanine 0.051 1.66
18. Isoleucine 0.047 1.72
19. Leucine 0.03 1.7
20. Lysine 0.028 1.66
21. Hydroxyproline 0.021 4.13
22. Sarcosine 0.026 1.15
23. Proline 0.021 4.36
4/19/2017
Agilent Confidential
15
Retention time and area RSD precision for AA_ 4.6 x 100 mm column_1000pmol (six replicates)
Amino Acids RT RSD
(%)
Area RS0.025D
(%)
1. Aspartic acid 0.151 2.60
2. Glutamic acid 0.512 2.19
3. Asparagine 0.124 2.13
4. Serine 0.114 1.74
5. Glutamine 0.092 1.8
6. Histidine 0.077 1.39
7. Glycine 0.068 1.48
8. Threonine 0.059 2.26
9. Arginine 0.027 1.66
10. Alanine 0.031 1.87
11. Tyrosine 0.034 2.04
Amino Acids RT RSD
(%)
Area RSD (%)
12. Cystine 0.030 2.22
13. Valine
0.025 2.4
14. Methionine
0.025 1.78
15. Norvaline 0.019 1.77
16. Tryptophan 0.024 2.03
17. Phenylalanine 0.027 1.98
18. Isoleucine 0.025 2.17
19. Leucine 0.020 1.81
20. Lysine 0.022 2
21. Hydroxyproline 0.014 3.14
22. Sarcosine 0.015 5.91
23. Proline 0.011 2.58
4/19/2017
Agilent Confidential
16
System suitability as per European Pharmacopoeia (Ph.Eur)
The European Pharmacacopoeia (Ph. Eur.) defines requirements for the
qualitative and quantitative composition of amino acids and mixtures of
amino acids. The requirements for allowed impurities are also defined.
Manufacturers of amino acids are legally bound to prove that their amino
acids meet these specifications before they can distribute their products in
Europe.
Leucine (Leu) is a branched-chain α-amino acid and is produced by the
fermentation process. During this process, isoleucine can be produced as a
by-product. The European Pharmacopoeia states that leucine and
isoleucine should have a resolution of not less than 1.5
Leucine Isoleucine
≥ 1.5
Ref: Ph.Eur.9.0 (2.2.56) Amino Acid Analysis
4/19/2017
Agilent Confidential
17
System suitability testing using AdvanceBio AAA columns and AA standards.
System Suitability Agilent AdvanceBio
AAA, C18, 4.6 × 100
mm, 2.7 μm
Agilent AdvanceBio
AAA, C18, 3 × 100 mm,
2.7 μm
Resolution between
Leucine and
Isoleucine
(≥1.5)
4.5
4.6
4/19/2017
Agilent Confidential
18
Linearity, LOD and LOQ
As an example Extended amino acid (EAA) stock solution, asparagine,
glutamine, and tryptophan were used for linearity, LOD and LOQ determination.
Linearity was evaluated up to 1000pmol.
The detection limit (LOD) and quantification limit (LOQ) were estimated from the
calibration function. LOD and LOQ were calculated as 3(SD(a)/b) and
10(SD(a)/b), respectively, where SD(a) is the standard deviation of the intercept
and b is the slope of the calibration function.
4/19/2017
Agilent Confidential
19
Linearity, LOD and LOQ
min 4 6 8
mAU
0
20
40
60
80
100
Aspara
gin
e
Glu
tam
ine
Try
pto
phan min 2.4 2.6 2.8
mAU
0
20
40
60
80
100
min 3.2 3.4 3.6
mAU
0
10
20
30
40
50
60
min 8.2 8.4 8.6
mAU
0
10
20
30
40
50
60
70
Asparagine Glutamine Tryptophan
4/19/2017
Agilent Confidential
20
y = 2.0654x + 2.6479R² = 0.9993
0
500
1000
1500
2000
2500
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Aspargine
y = 1.0107x + 0.3716R² = 0.9997
0
200
400
600
800
1000
1200
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Glutamine
y = 1.6446x + 6.0767R² = 0.9999
0
200
400
600
800
1000
1200
1400
1600
1800
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Tryptophan
Linearity curves for Asn, Gln and Trp
4/19/2017
Agilent Confidential
21
LOD and LOQ
Concentration (pmol) S/N ratio
Asparagine
0.9 (LOD) 5.3
1.9 (LOQ) 10.8
Glutamine
0.9 (LOD) 3.0
3.8 (LOQ) 13.8
Tryptophan
0.9 (LOD) 4.5
3.8 (LOQ) 20.5
LOD, LOQ and S/N ratio (n=3)
S/N >3.0 = LOD
S/N> 10 = LOQ
4/19/2017
Agilent Confidential
22
Cell Media Media and protein hydrolysate standard
1. Minimum Essential Medium Eagle (MEM) M4655 – L-Arginine, L-Cystine, L-
Glutamine, L-Histidine, L-Isoleucine, L- Leucine, L-Lysine, L-Methionine, L- Phenylalanine,
L-Threonine, L-Tryptophan, L- Tyrosine and L-Valine.
2. Non-Essential Amino Acid (NEAA) Cell Culture Supplement M7145 – L-Alanine,
L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline and L-Serine
3. RPMI 1640 R0083 - L-Arginine, L-Asparagine, L-Cystine, Glycine, L-Histidine, Hydroxy-
L-Proline, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-
Serine, L-Threonine, L-Tryptophan, L-Tyrosine and L-Valine
4. Protein Hydrosylate Standard - L-Aspartic acid, L-Glutamic acid, L-Serine, L-
Histidine, Glycine, L-Threonine, L-Arginine, L-Alanine, L- Tyrosine, L-Cystine, L-Valine, L-
Methionine, L-Phenylalanine, L-Isoleucine, L- Leucine, L-Lysine and L-Proline
4/19/2017
Agilent Confidential
23
min 2 3 4 5 6 7 8 9
mAU
100
50
0
50
100
150
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
e
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Glu
tam
ic a
cid
Glu
tam
ine
His
tid
ine
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e Cys
tin
e
Val
ine
Me
thio
nin
e
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
MEM Eagle , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
AAA of Cell Media – MEM L-Arginine, L-Cystine, L-Glutamine, L-Histidine, L-Isoleucine, L- Leucine, L-Lysine, L-Methionine, L- Phenylalanine, L-Threonine, L-
Tryptophan, L- Tyrosine and L-Valine, L-Glutamic acid
4/19/2017
Agilent Confidential
24
AAA of Cell Media – NEAA cell culture supplement L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline and L-Serine
min 2 4 6 8 10 12
mAU
80
60
40
20
0
20
40
60
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
H
isti
din
e
Gly
cin
e
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Asp
arti
c ac
id
Asp
arti
c ac
id
Ala
nin
e
Asp
arag
ine
Glu
tam
ic a
cid
Gly
cin
e
Pro
lin
e Se
rin
e
NEAA cell culture supplement , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
4/19/2017
Agilent Confidential
25
AAA of Cell Media – RPMI 1640 L-Arginine, L-Glutamic acid, L-Asparagine, L-Cystine, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,
L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine and L-Valine
min 2 4 6 8 10 12
mAU
150
100
50
0
50
100
150
200
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
H
isti
din
e
Gly
cin
e
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Tryp
top
han
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Asp
arti
c ac
id
Pro
lin
e
Arg
inin
e
Asp
arag
ine
Cys
tin
e
Gly
cin
e
His
tid
ine
Hyd
roxy
pro
lin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Me
thio
nin
e
Ph
en
ylal
anin
e Se
rin
e
Thre
on
ine
Tryp
top
han
Tyro
sin
e
Val
ine
Glu
tam
ic a
cid
Asp
arti
c ac
id
RPMI 1640 Media , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
4/19/2017
Agilent Confidential
26
Separation of Protein Hydrosylate Standard
Baseline resolution of isoleucine and leucine (Rs = 4.35) meeting the
regulatory requirements for these components.
min 2 4 6 8 10 12
mAU
0
100
200
300
400
500 DAD1 A, Sig=338,10 Ref=390,20,
Aspart
ic a
cid
Glu
tam
ic a
cid
Serine
Try
osin
e
His
tidin
e Gly
cin
e
Thre
onin
e
Ala
nin
e
Cyste
ine
Valin
e
Meth
ionin
e
Phenyla
lanin
e
Isole
ucin
e
Leucin
e
Lysin
e
. P
rolin
e
Rs = 4.35
Arg
inin
e
4/19/2017
Agilent Confidential
27
Separation of amino acid and internal standards on three lots of AdvanceBio AAA, 4.6 × 100 mm, 2.7 μm columns
Lot 2
Lot 3
min 2 4 6 8 10 12
mAU
0
20
40
60
80
min 2 4 6 8 10 12
mAU
0
20
40
60
80
min 2 4 6 8 10 12
mAU
0
20
40
60
80
Lot 1
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Tryp
top
han
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Asp
arti
c ac
id
Thre
on
ine
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Separation of amino acid and internal standards on three lots of AdvanceBio AAA,
3 × 100 mm, 2.7 μm columns
Lot 1
Lot 2
Lot 3
min 2 4 6 8 10 12
mAU
0
20
40
60
80
100
120
min 2 4 6 8 10 12
mAU
0
20
40
60
80
100
120
min 2 4 6 8 10 12
mAU
0
20
40
60
80
100
120
wjl
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Tryp
top
han
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Asp
arti
c ac
id
Thre
on
ine
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Value Propositions – Technical Value
For Biopharma customers using Reverse phase for high resolution separation of primary and
secondary amino acid from protein hydrolysate/CCM using pre column derivatization.
Who Require automation for seamless derivatization combined with rugged column chemistry
for high pH applications
Our Product AdvanceBio AAA Reagent kit + AdvanceBio AAA columns with Agilent Analytical LCs
Provides A proven OPA/FMOC online derivatization for primary and secondary amino acid and
separation on AdvanceBio AAA column which provides excellent peak shape and
resolution for all 23 amino acid using UV and more sensitive FLD detection
Our solution A complete solution for amino acid analysis - combining efficient pre-column
derivatization using autosampler, proven OPA/FMOC chemistry for derivatization and
AdvanceBio AAA superficially porous columns.
4/19/2017
Agilent Confidential
30
Key Value
• Up to twice the column lifetime compared to standard silica
- Specially modified Poroshell particle for high pH stability
- 2µm inlet frit for less chance of clogging
• Columns batch tested with AA standards to ensure quality and performance
• Established methods and workflow utilizes unique features of Agilent Autosampler
- Automated pre-column derivatization for faster sample prep with less variability than manual
- Complete method in under 20 minutes!
• Reagents and standards in one kit under one part number
- ease of ordering for start up
- Components available separately
• Longer column life + faster more consistent derivatization + higher quality = more samples per
column+ more reliable data = cost savings to our customer!
4/19/2017
Agilent Confidential
31
Tips and Tricks
Maintenance
• Replace derivatization reagent, borate buffer, amino acid standard daily.
• Recalibrate for retention times and response factors daily.
• Check column and guard column performance by following specs (Rs for
2 pairs of AA).
• Replace mobile phase A and B with fresh ones every alternate day.
• Exchange guard column if high back pressure develops.
4/19/2017
Agilent Confidential
32
Tips and Tricks
Poor chromatographic resolution
• In all cases, use the low-volume heat exchanger with short tubing to minimize
extra column volume.
• Damaged guard or analytical column
• Cell culture media does not require any sample preparation, however
appropriate dilutions have to be made to suit detector response.
Low intensity chromatogram
• OPA/FMOC reagent deteriorated.
Column storage
• For long term and short term always store the column in mobile phase B.
Never leave the column in mobile phase A even if its overnight.
4/19/2017
Agilent Confidential
33
Summary
• We demonstrate the utility of AdvanceBio AAA kit and AdvanceBio AA column for the
automated online derivatization and separation of amino acids using OPA/FMOC
chemistries.
• Area and RT precision of the method were excellent and met the system suitability
requirement.
• Linearity curves with ten standard concentrations of three amino acids, ranging from
0.9pmol to 1nmol, had excellent coefficient of linearity values, indicating that the method
was quantitative and accurate.
• The LOD and LOQ for the amino acids were 0.9pmol and 3.8 pmol respectively, indicating
that the method was sensitive.
• This method was able to separate and detect, amino acids from cell culture media and
protein hydrolysate standard. The amino acid composition determined using this method
correlated very well with their theoretical composition.
4/19/2017
Agilent Confidential
34
• Product Flyer: 5991-7696EN
• Poster (WCBP 2017): 5991-7877EN
• Application Note: 5991-7693EN
• Detailed “How To Guide” 5991-7694EN
4/19/2017
Agilent Confidential
35
Collaterals
Questions?
Thanks for your attention!