Applications of 2D-LC in Pharmaceutical Analysis · PDF fileApplications of 2D-LC in Pharmaceutical Analysis ... Impurities with tox coverage, ... Chromatographic profile of sample

Applications of 2D-LC in Pharmaceutical Analysis

C. J. Venkatramani, Genentech, USA

June 22nd, 2016 - HPLC 2016 - Agilent Lunch Seminar

©2016Genentech,Inc. 1

Agenda Slide 2

•  Business driver – why 2D-LC? •  Flavors of Two-Dimensional Chromatography •  Two-Dimensional Liquid Chromatography •  Application of 2D-LC in pharmaceutical analysis •  Acknowledgements

©2016Genentech,Inc.

Pharmaceutical Industry

Highly regulated industry Deliver safe and efficacious medications patients DS Specifications: Appearance, Identity, Assay and

Related Substances, Residual Solvents, Water Content, Heavy Metals and Specified Metals ….


Slide 3


Slide 4

ICH Q3A:

Reporting threshold > 0.05%

Identification threshold ≥ 0.10% or max 1.0 mg/day

Qualification threshold ≥ 0.15% or max 1.0 mg/day)

Exceptions: Impurities with tox coverage, metabolites Genotoxic impurities (M7):

Limited by max daily dose & duration of exposure Usually low parts per million

Impurities in New Drug Substances

Drug substance synthetic scheme (Hypothetical)


Slide 5

Regulatory Starting Material (RSM1)

Drug Substance

O

OH

F F

ClO

N

HN

NH

O

NH

R

O

O

O

O

R R'

Cl

5-chloro-2,4-difluorobenzoic acidRSM 1

IntermediateStage 2

Drug Substance

O

OH

F F

Cl

O

OHF

F

Cl

O

OH

F

F

Cl O

OHF

F Cl

O

OH

FF

ClO

OH

F

F

Cl

O

OH

F

F

Cl

O

OHF

F

Cl

O

OH

F

F

Cl

O

OHF

F

Cl

O

OH

FF

Cl

O

OHF

F

Cl

O

OHF

FCl

O

OH

F

F

Cl

O

OHF

F

Cl

O

OH

F

F

Cl

O

OH

F

F

Cl

O

OH

F F

Cl

O

OH

F

F

Cl

O

OH

F

F

Cl

O

OH

F

F

Cl

O

OH

FF

Cl O

OHF

F

Cl

O

OH

F

F

ClO

O

OHF

F

Cl

O

OH

F

F

Cl

O

OH

F

F

Cl

O

OH

F

F Cl

Areaofinterest

API

Organic impurities from reg. starting material


Slide 6

Isomers are likely to react and produce associated API impurities which will be difficult to purge, analyze

Slide7 ©2016Genentech,Inc.

Organic impurities in SM pharmaceuticals Strategy for impurity characterization Screen columns of different selectivity, pH’s, peak tracking Demonstrate specificity - SM’s, intermediates, potential imps

Demonstrate method is stability indicating with stressed samples - acid, base, peroxide, heat, humidity, light

Relies on DAD and MS for detection - limiting factor Peaks eluting around main component have similar

UV spectra - Limits DAD Isomers - Limits MS

Potential Solution: Two-dimensional Chromatography

Slide 7

Flavors of 2D-LC


Slide9

Flavors of 2D Chromatography

Heart-Cutting 2D-LC Part of primary column eluent sampled into secondary column Easy to operate, adequate for most applications – camera

Comprehensive 2D-LC

Entire primary column eluent sampled into secondary column Challenging, detailed analysis- camcorder

Pseudo-comprehensive 2D-LC

Comprehensive separation of select region of primary column eluent (Targeted analysis) Moderate level of difficulty, adequate for most applications – smart phone


Slide 9

\D\DHeart-cutting 2D-LC

Sec.col.Separa3on(Chirobio3cT)

NH2

O OH

d-phenylalanine

NH2

O OH

l-phenylalanine

1

2 3 4 5 6

0

20

40

60

80

100

120

Retention Time (min)

Det

. Res

pons

e (m

AU

)

Primarycol.Separa3on(ODS-AQ)


C. J. Venkatramani, Larry Wigman, Kavita Mistry and Nichols Chetwyn, Simultaneous, sequential quantitative aciral-chiral analysis by two-dimensional liquid chromatography, Journal of Separation Science, Vol. 35, p1748, 2012

Slide 10

Slide 11

Primary

Chromatogram

Comprehensive 2D-LC

PrimaryColum

nReten3on(minutes)

5.00 5.50

6.00 6.50

7.00 7.50

8.00

DetectorResponse(mAU)

-200 0 200 400 600 800

1000 Secondary

Chromatograms 2D Contour Plots

Sec. Ret. (sec)

Prim

ary Ret. (m

in)


Slide 11

Primary

Chromatogram

PrimaryColum

nReten3on(minutes)

5.00 5.50

6.00 6.50

7.00 7.50

8.00

DetectorResponse(mAU)

-200 0 200 400 600 800

1000 Secondary

Chromatograms 2D Contour Plots

Sec. Ret. (sec)

Prim

ary Ret. (m

in)


Slide 12 Pseudo-comprehensive 2D-LC (Targeted analysis)

Agilent – Multiple heart-cutting

Case study: 2D-LC-MS method development strategy for complex, high molecular weight compound


Analytical challenges Slide 14


Extremely complex synthesis involving multiple steps

Lot to lot variability requiring iterative method development

Some of the intermediates are extremely reactive and toxic Molecular weights is usually very high Qualitative and quantitative analysis of potential impurities is critical as it could have significant bearing on the downstream process and long term stability

Chromatographic conditions Slide 15

©2016Genentech,Inc. IlaPatel,Genentech

Parameter Value

Column Ace, Excel 2 PFP C18, 150 mm x 3.0 mm, 2.0um

Wavelength 205 nm

Oven Temperature 40 °C

Flow Rate 0.5 mL/min.

Injection Volume 5 µL

Mobile Phase A 0.05% phosphoric acid in water

Mobile Phase B 0.05% phosphoric acid in ACN

Gradient program

Time (min) A% B% 0.0 95 5 8.0 60 40 23.0 47 53 28.0 5 95 30.0 5 95 30.1 95 5 37.0 95 5

Run Time 37 min

Diluent 50/50 (v/v) Acetonitrile / Water

Chromatographic profile of sample Slide 16

©2016Genentech,Inc. IlaPatel,Genentech

Strategy for 2D-LC analysis Slide 17


Modify primary column gradient to elute component of interest within 5 to 15 minutes Evaluate complementary phases in the secondary dimension

SB-CN, Primesep-B, diamond hydride phase, SB-Phenyl, SB-Aq…

Re-assess original method using most complementary phase in the secondary dimension

Modified expt. conditions – primary column Slide 18


Pictorial representation of expt. set-up Slide 19


Traceinred:PrimarycolumngradientTraceinblue:Secondarycolumngradient

Frac3onstransferredtosec.column

1stgradient Lastgradient

Experimental conditions – secondary column Slide 20


*Frac3ons1to5areparkedinDeckA,analyzedinreverseorder(5,4,3….)*Frac3ons6to10parkedinDeckB*Frac3ons6through10inDeckBisanalyzedinrevereseorder(10,9,8...)followingDeckAanalysis*Trappingandanalysiscanrepeatedthroughoutthechromatogram

High resolution sampling 2D-LC (Heart-cutting) Slide 21


Park

Analysis

Park

Analysis

Frac3o

n#6

Frac3o

n#7

Frac3o

n#8

Frac3o

n#9

Frac3o

n#10

High-resolution sampling 2D-LC Slide 22


PrimaryColumnSepara3on SecColumnSepara3onSecColumnSepara3on

Cut2

Cut3

Cut4

Cut5

Cut6

Cut7

Cut8

Cut9

A B

A B

A B

AB C

C

C

C

Composite picture of 2D-LC separation (main comp) Slide 23


Stackplotofseccolumnsepara3onshowingpoten3alco-elu3onintheprimarydimension

ImpA

ImpB

ImpC

Finalintermediate

??

Lot#1

Stack plot of 2nd dimension separation Slide 24


Maincomponent

MStraceofsamplelot

UVtraceofsamplelotat260nm

*Co-elu3ngimpuri3esfromtheprimarycolumnareresolvedinthesecondary,complementarySB-CNcolumn*Impuri3eswerenotdetectedbyMS

2D-LC detection: UV v/s MS Slide 25


Cut#5MS

UV

Cut#6MS

UV

*Co-elu3ngimpuri3esfromtheprimarycolumnareresolvedinthesecondary,complementarySB-CNcolumn*Impuri3eswerenotdetectedbyMS

2D-LC detection: UV v/s MS Slide 26


A B

Impuri3esAandBobservedinfrac3ons2to5acrossthemainpeak

Zones of co-elution in the 1st dimension Slide 27


C

ImpurityCobservedinfrac3ons5to9acrossthemainpeak

Zones of co-elution in the 1st dimension Slide 28


Primarycolumnsepara3on

8.31min8.85min

Seccolumnsepara3on(UV)

Assessment of 1st dimension for potential co-elusion Slide 29


10.95min11.03min11.11min



Selective assessment of 1st dimension (co-elusion) Slide 30


11.45minMainComp





Seccolumnsepara3on13.49min13.69min13.89min



(4)13.49min13.69min13.89min

(1)8.31min8.85min

(2)10.95min11.03min11.11min

(3)11.45minMainComp

12 3 4



Basedon2D-LC-MSanalysisofsample,mul3plecomponentsco-eluteintheprimarydimension,severalofthese

componentsarepar3allyresolvedinthesecondarySB-CNcolumn



Lot#2

ImpA ImpA

Finalintermediate

Stackplotofsecondarycolumnsepara3onshowingpoten3alco-elu3oninprimarydimension

Sec. column separation of lot 2 (main component) Slide 35


Isocratic separation in secondary SB-CN column


AB

C

Lot1,HPLCUVat260nm

Secondarycolumnsepara3onisocra3csepara3on(Water:ACN:FA48:52:0.05)

Sec. column separation - Isocratic Slide 37

Finalintermediate



AB

Lot2,HPLCUVat260nm



Quantitation by 2D-LC


Secondarycolumnsepara3on(min)

Secondarycolumnpeakintegra3on

Peak ID & quantification in 2nd dimension Slide 40


Realcomponentwillshowsupinmul3ple,sequen3alchromatogramsconfirmingitspresenceinsample

Quantitation: multiple heart cutting 2D-LC Slide 41


*Rela3vePA’softheimpuri3esarecomparablebetweenrunslow%RSD(<10%)forpeaksintheLOQrange

*Lot#1hasanaddi3onalimpuritycomparedtolot1*%PAisthelevelofimpuri3esco-elu3nginthemaincomponent

Lot1

Lot2

RetentionTime Run#1 Run#2 Run#3 Average StdDev %RSD %PeakArea(min)0.9 7.3 7.9 7.5 7.6 0.30 3.98 0.060.99 12.4 14.0 13.9 13.4 0.92 6.83 0.111.21 32.045 30.935 30.998 31.3 0.62 1.99 0.261.42 11922.2 11875.9 11907.3 11901.8 23.65 0.20 99.56

Quantitative analysis of co-eluting imps (main comp) Slide 42


Blank@300nm

Sample@300nm

Blank@260n

m

Sample@260nm

Comparison of blank and sample at 260 and 300 nm Slide 43


C


AB

Lot2,HPLCUVat260nm

Effec3vereten3onspace



Hi-speed separation in secondary column


SecondaryColumnSepara3on(2.1mmx3cmx1.8uMRRHDCN)

Cut2

Cut3

Cut4

Cut5

Cut6

Cut7

Cut8

Cut9

Cut10

A/B

A/B

A/B

C

C

C

C

C

Sec. column separation on RRHD SB-CN column Slide 46


Assessment of potential co-elution in original method


Lot1,260nmOriginalmethodPhosphoricacidsystem

Lot1,260nmCut3

A B

FinalInt Lot1,260nmCut6

C?

FinalInt

Sec. column separation – SB-CN Slide 48


Lot1,260nmOriginalmethodPhosphoricacidsystem

Lot1,260nm

Lot1,300nm

Deg1

Sec. column separation – SB-CN Slide 49

©2016Genentech,Inc.Residuallevelofnewdegrada3onproductobservedinagedsample

ImpCresolvedfrommaincomponentinthereleasemethod

Analysis of sample using Primesep-B column in secondary dimension


Secondarycolumnsepara3onona2cmPrimesep-Bguardcolumn

Secondarycolumnsepara3on

Primarycolumnsepara3on LC-UV@260nm

C

HPLC-UV

Sec. column separation – PS-B Slide 51


Analysis of sample using diamond hydride column in secondary dimension


Secondarycolumnsepara3on

Primarycolumnsepara3on LC-UV@260nm

Secondarycolumnsepara3ononaDiamondHydridecolumn(5cmx4mmx4micron)

FinalIntermediate

HPLC-UVC

Sec. column separation – DH Slide 53


Analysis of sample using SB-Aq column in secondary dimension


Seccolumnsepara3ononaSB-AQcolumn,2.1mmx30mm,1.8uM

CLot1

Sec. column separation – SB-Aq Slide 55


Slide56

Conclusions

Successfully demonstrated the applications of 2D-LC in resolving residual, co-eluting impurities in the midst of main component. Successfully demonstrated the capability of hi-resolution 2D-LC in quantitative analysis of co-eluting impurities

SB-CN offered most complementary separation in the secondary dimension. Commercial systems have extended the capabilities of 2D-LC from being a research tool in select laboratories to real world applications

Multiple heart cutting, flexibility …...


Slide 56

Acknowledgments

AgilentTechnologies•  LisaZang•  MichaelYap•  JenniferThompson•  RussellBencomo•  MichaelFrank•  BrinkerGildersleeve

Genentech•  JacobKay•  IlaPatel•  MohammadAl-Sayah•  LarryWigman•  PeterYehl


Slide 57

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Applications of 2D-LC in Pharmaceutical Analysis · PDF fileApplications of 2D-LC in Pharmaceutical Analysis ... Impurities with tox coverage, ... Chromatographic profile of sample