Applications of 2D-LC in Pharmaceutical Analysis

Transcription

1 Applications of 2D-LC in Pharmaceutical Analysis C. J. Venkatramani, Genentech, USA June 22 nd, HPLC Agilent Lunch Seminar 1

2 Agenda Slide 2 Business driver why 2D-LC? lavors of Two-Dimensional Chromatography Two-Dimensional Liquid Chromatography Application of 2D-LC in pharmaceutical analysis Acknowledgements

3 Pharmaceutical Industry Slide 3 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.

4 Impurities in New Drug Substances 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

5 Drug substance synthetic scheme (Hypothetical) Slide 5 R N H Cl H Intermediate Stage 2 Cl N H N N H R R' 5-chloro-2,4-difluorobenzoic acid RSM 1 Regulatory Starting Material (RSM1) Drug Substance Drug Substance

6 rganic impurities from reg. starting material Slide 6 H Cl H API H Cl Cl H Cl H H Cl H Cl H Cl H Cl Cl H Cl Cl H H H Cl H Cl H Area of interest H Cl Cl H Cl H Cl Cl Cl H H H Cl H Cl H Cl Cl Isomers are likely to Cl react and produce associated API H H H H H impurities Cl which Cl Cl will be difficult Clto purge, analyze

7 rganic Strategy for impurities impurity in characterization SM pharmaceuticals Slide 7 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

8 lavors of 2D-LC 8

9 lavors of 2D Chromatography Slide 9 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

10 \ D Heart-cutting 2D-LC \ D Slide H Det. Response (mau) Sec. col. Separa3on (Chirobio3c T) l-phenylalanine H NH 2 d-phenylalanine NH 2 0 Primary col. Separa3on (DS-AQ) Retention Time (min) 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

11 Comprehensive 2D-LC Slide 11 Detector Response (mau) Primary Column Reten3on (minutes) Primary Ret. (min) Sec. Ret. (sec) Primary Chromatogram Secondary Chromatograms 2D Contour Plots Slide 11

12 Pseudo-comprehensive 2D-LC (Targeted analysis) Slide 12 Detector Response (mau) Primary Column Reten3on (minutes) Primary Ret. (min) Sec. Ret. (sec) Primary Chromatogram Secondary Chromatograms 2D Contour Plots Agilent Multiple heart-cutting

13 Case study: 2D-LC-MS method development strategy for complex, high molecular weight compound 13

14 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

15 Chromatographic conditions Slide 15 Parameter Column Wavelength Value Ace, Excel 2 PP C18, 150 mm x 3.0 mm, 2.0um 205 nm ven Temperature 40 C low Rate 0.5 ml/min. Injection Volume 5 µl Mobile Phase A Mobile Phase B Gradient program Run Time Diluent 0.05% phosphoric acid in water 0.05% phosphoric acid in ACN 37 min Time (min) A% B% /50 (v/v) Acetonitrile / Water Ila Patel, Genentech

16 Chromatographic profile of sample Slide 16 Ila Patel, Genentech

17 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

18 Modified expt. conditions primary column Slide 18

19 Pictorial representation of expt. set-up Slide 19 1 st gradient Last gradient rac3ons transferred to sec. column Trace in red: Primary column gradient Trace in blue: Secondary column gradient

20 Experimental conditions secondary column Slide 20

21 High resolution sampling 2D-LC (Heart-cutting) Slide 21 Park Analysis Park Analysis *rac3ons 1 to 5 are parked in Deck A, analyzed in reverse order (5, 4, 3.) *rac3ons 6 to 10 parked in Deck B *rac3ons 6 through 10 in Deck B is analyzed in reverese order (10, 9, 8...) following Deck A analysis *Trapping and analysis can repeated throughout 2016 Genentech, Inc. the chromatogram

22 High-resolution sampling 2D-LC Slide 22 rac3on #10 rac3on #9 rac3on #8 rac3on #7 rac3on #6

23 Composite picture of 2D-LC separation (main comp) Slide 23 Cut 2 Cut 6 C A B Cut 3 A B Cut 7 C Cut 4 Cut 8 A B C Cut 5 Cut 9 A B C Sec Column Separa3on Primary Column Separa3on Sec Column Separa3on

24 Stack plot of 2 nd dimension separation Slide 24 Lot #1 inal intermediate Imp C Imp A Imp B?? Main component Stack plot of sec column separa3on showing poten3al co-elu3on in the primary dimension

25 2D-LC detection: UV v/s MS Slide 25 MS trace of sample lot UV trace of sample lot at 260 nm *Co-elu3ng impuri3es from the primary column are resolved in the secondary, complementary SB-CN column *Impuri3es were not detected by MS

26 2D-LC detection: UV v/s MS Slide 26 Cut #5 MS Cut #6 MS UV UV *Co-elu3ng impuri3es from the primary column are resolved in the secondary, complementary SB-CN column *Impuri3es were not detected by MS

27 Zones of co-elution in the 1 st dimension Slide 27 A B Impuri3es A and B observed in frac3ons 2 to 5 across the main peak

28 Zones of co-elution in the 1 st dimension Slide 28 C Impurity C observed in frac3ons 5 to 9 across the main peak

29 Assessment of 1 st dimension for potential co-elusion Slide 29 Sec column separa3on (UV) 8.31 min 8.85 min Primary column 2016 Genentech, separa3on Inc.

30 Selective assessment of 1 st dimension (co-elusion) Slide 30 Sec column separa3on (UV) min min min Primary column 2016 Genentech, separa3on Inc.

31 Selective assessment of 1 st dimension (co-elusion) Slide 31 Sec column separa3on (UV) min Main Comp Primary column 2016 Genentech, separa3on Inc.

32 Selective assessment of 1 st dimension (co-elusion) Slide 32 Sec column separa3on min min min

33 Selective assessment of 1 st dimension (co-elusion) Slide 33 (2) min min min (3) min Main Comp (4) min min min (1) 8.31 min 8.85 min

34 Selective assessment of 1 st dimension (co-elusion) Slide 34 Based on 2D-LC-MS analysis of sample, mul3ple components co-elute in the primary dimension, several of these components are par3ally resolved in the secondary SB-CN column

35 Sec. column separation of lot 2 (main component) Slide 35 Lot #2 inal intermediate Imp A Imp A Stack plot of secondary column separa3on showing poten3al co-elu3on in primary dimension

36 Isocratic separation in secondary SB-CN column 36

37 Sec. column separation - Isocratic Slide 37 Lot 1, HPLC UV at 260 nm A B inal intermediate C Secondary column separa3on isocra3c separa3on (Water:ACN:A 48:52:0.05)

38 Sec. column separation - Isocratic Slide 38 Lot 2, HPLC UV at 260 nm A B Secondary column separa3on isocra3c separa3on (Water:ACN:A 48:52:0.05)

39 Quantitation by 2D-LC 39

40 Peak ID & quantification in 2 nd dimension Slide 40 Secondary column peak integra3on Secondary column 2016 Genentech, separa3on Inc. (min)

41 Quantitation: multiple heart cutting 2D-LC Slide 41 Real component will shows up in mul3ple, sequen3al chromatograms confirming its presence in sample

42 Quantitative analysis of co-eluting imps (main comp) Slide 42 Lot 1 Retention Time Run#1 Run#2 Run#3 Average Std Dev %RSD %Peak Area (min) Lot 2 *Rela3ve PA s of the impuri3es are comparable between runs low %RSD (< 10%) for peaks in the LQ range *Lot#1 has an addi3onal impurity compared to lot 1 *%PA is the level of impuri3es co-elu3ng 2016 Genentech, in the Inc. main component

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

44 Sec. column separation - Isocratic Slide 44 Lot 2, HPLC UV at 260 nm A B Effec3ve reten3on space Secondary column separa3on isocra3c separa3on (Water:ACN:A 48:52:0.05)

45 Hi-speed separation in secondary column 45

46 Sec. column separation on RRHD SB-CN column Slide 46 Cut 2 Cut 5 Cut 8 A/B C C A/B Cut 3 C Cut 6 Cut 9 C A/B Cut 4 C Cut 7 Cut 10 Secondary Column Separa3on (2.1 mm x 3 cm x 1.8 um RRHD CN)

47 Assessment of potential co-elution in original method 47

48 Sec. column separation SB-CN Slide 48 Lot 1, 260 nm Cut 3 A B inal Int Lot 1, 260 nm Cut 6 inal Int C? Lot 1, 260 nm riginal method Phosphoric acid system

49 Sec. column separation SB-CN Slide 49 Deg 1 Lot 1, 260 nm Lot 1, 300 nm Lot 1, 260 nm riginal method Phosphoric acid system Residual level of new degrada3on product observed in aged sample Imp C resolved from main component in the release method

50 Analysis of sample using Primesep-B column in secondary dimension 50

51 Sec. column separation PS-B Slide 51 Primary column separa3on 260 nm HPLC-UV Secondary column separa3on C Secondary column separa3on on a 2 cm Primesep-B guard column

52 Analysis of sample using diamond hydride column in secondary dimension 52

53 Sec. column separation DH Slide 53 Primary column separa3on 260 nm HPLC-UV C inal Intermediate Secondary column separa3on Secondary column separa3on on a Diamond Hydride column (5 cm x 4mm x 4 micron)

54 Analysis of sample using SB-Aq column in secondary dimension 54

55 Sec. column separation SB-Aq Slide 55 C Lot 1 Sec column separa3on on a SB-AQ column, 2.1 mm x 30 mm, 1.8 um

56 Conclusions Slide 56 Successfully demonstrated the applications of 2D-LC in resolving residual, coeluting 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

57 Acknowledgments Slide 57 Genentech Jacob Kay Ila Patel Mohammad Al-Sayah Larry Wigman Peter Yehl Agilent Technologies Lisa Zang Michael Yap Jennifer Thompson Russell Bencomo Michael rank Brinker Gildersleeve