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


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

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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

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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

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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

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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.

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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


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

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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

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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

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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

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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

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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

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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

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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

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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.

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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


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


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


Linearity for Tryptophan

Linearity curves for Asn, Gln and Trp

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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


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


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

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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,


4/19/2017


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,


4/19/2017


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


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.

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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!

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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.

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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.

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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


34

• Product Flyer: 5991-7696EN

• Poster (WCBP 2017): 5991-7877EN

• Application Note: 5991-7693EN

• Detailed “How To Guide” 5991-7694EN

4/19/2017


35

Collaterals

Questions?

Thanks for your attention!

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