Drug Degradation Mechanisms: Why Are They Important?

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Drug Degradation Mechanisms: Why Are They Important? Steven W. Baertschi, Ph.D. Baertschi Consulting, LLC http://baertschiconsulting.

1 Drug Degradation Mechanisms: Why Are They Important? Steven W. Baertschi, Ph.D. Baertschi Consulting, LLC Sept 25-27, 2016 Science of Stability Conference Newark, NJ 1 1

2 Introduction I. Drug Degradation Mechanisms II. Can t we just control empirically? Do we really need to develop mechanistic understanding? Is such understanding really a necessary part of Quality-By- Design? Five Case Studies 1. Unexplained peaks in a chromatogram 2. Unexpected racemization of an API 3 and 4. Non-homogeneous color change in API upon storage 5. Severe unexplained loss of API upon storage (mass balance) III. Conclusions 2

3 3 Better Understanding for Better Control Through Chemistry

4 What do we mean by mechanism? In chemistry, a reaction mechanism is the step by step sequence of elementary reactions by which overall chemical change occurs. For the purpose of this presentation Mechanism= the initiating step and the subsequent cascade Typically, the initiating step is a potential control point. The subsequent cascade is a potential control point, diversion- or stop-point. 4

5 Case Study #1: Unexpected New Peaks in Chromatogram 5

6 Optimum Conditions for Litronesib based on Column Screening Strategy PARAMETER SETTING Column Waters X-Bridge C18, 4.6 x 75 mm, 2.5 um Flow rate 1.0 ml/min Gradient profile Time (minutes) % A (0.05% Ammonium Hydroxide) Autosampler temperature 5ºC Detection wavelength 290 nm Column temperature 30ºC Injection volume 10 μl % B (Acetontrile) Above conditions showed excellent selectivity and peak shape for Compound A and its key impurities. During testing of the method for use in process development new significant impurities (>0.5%) were noted. 6

7 Case Study #1: Unexpected New Peaks in Chromatogram Unexpected New Peaks 7

8 Observations in the newly developed HPLC conditions New peaks at levels varied (0.7 to 1.0% each) for the same sample Not formed in diluent: Observed in samples prepared and injected immediately Varying levels on different X-Bridge C18 columns New Impurity peaks not seen in previous low ph conditions suggested peaks were artifactural - likely an on-column interaction 8

9 Initial Observation of the Impurities in Litronesib (HPLC UV chromatogram) Waters X-Bridge C18, 4.6 x 75 mm, 2.5 um Mobile Phase A- 0.05% NH 4 OH Mobile Phase B- Acetonitrile Acc. Mass MS = Parent + N,O - H 9

10 Identification of Structure Modification and Reaction to Synthesize Products were produced in nearly 100% yield after optimization of the amount of the mole ratio of compound A to NaNO 2 and ph to 1 with HCl 10

11 So how is Litronesib being N- nitrosylated on-column?? Where does the N=O come from? Acetonitrile? -- No (pardon the unavoidable pun) Ammonia? Yes! 15 N-labelling experiment showed that the nitrogen came came from ammonia. Griess assay showed positive formation of NOX eluting from the column 11

12 On-column reaction increases with column use 12 Relative total nitrosamine peak area percents (UV at 290 nm) for consecutive injections on the column

13 Reaction appears to occur at the head of the column (not in the frit) Used column Used Frit New column Used Frit Used column New Frit New column (New Frit) 13

14 X-Bridge C18 Separations with 50 um EDTA added to the mobile Phase-LC UV data mau Before and After EDTA added to Mobile Phase A Waters X-Bridge C18, 4.6 x 75 mm, 2.5 um Mobile Phase A- 0.05% NH 4 OH + 50 um EDTA *DAD1 A, Sig=290,16 Ref=400,10 (D:\DATA\EG5 2012\DM JAN13_1\DM_JAN_13_ \ D) *DAD1 A, Sig=290,16 Ref=400,10 (D:\DATA\EG5 2012\DM JAN13_1\DM_JAN_13_ \ D) *DAD1 A, Sig=290,16 Ref=400,10 (D:\DATA\EG5 2012\DM JAN13\DM_JAN_13_ \ D) Mobile Phase B- Acetonitrile 20 Reduces total nitrosamines peak area by ~ 10x 15 Standard Mobile Phase A 10 Standard Mobile Phase A w/ EDTA 5 Standard Mobile Phase A w/ EDTA min 14

15 Current Hypothesis for Mechanism of Nitrosamine formation Source of metal ion M+ is likely in all stainless steel inlet frits used in column hardware 2 Carcinogenesis vol.18 no.9 pp ,

16 So how is Litronesib being N-nitrosylated on-column?? Mechanistic insight: Ammonia is being oxidized to NOx by metals deposited on the head of the column Rxn occurs at high ph, under high pressure Rxn requires presence of acetonitrile, which likely acts as a ligand to some metal to potentiate the oxidation Control: Eliminate acetonitrile, or ammonia Add EDTA to mobile phase Non-metal frits 16

17 Case study #2: Unexpected racemization Liquid formulation found to be chirally-unstable at basic ph A chemical rationale for BASE catalysis was elusive Control achieved empirically (acidification, buffer change) 17

18 Predicted Acid-Catalyzed Racemization Pathway Ruled Out Scheme 1. Postulated acid-catalyzed racemization of (R)-litronesib. This pathway was excluded by the effect of ph on the rate of racemization. 18

19 No mechanistic insight scaffold risks? Since no mechanism could be developed, insight was sought to enable future scaffold improvements Experiments designed to probe mechanism 19

20 Proposed Mechanism Based on Experimental Data 20

21 Probing the Proposed Mechanism: Experiments with Two Analogs 21

22 22 Final Question: C-N or C-S cleavage?

23 Computational studies clearly implicate C-S cleavage Density functional theory calculations of the potential energy surface carried out at the M062X/6 31+G(d) level of theory (courtesy Mike Watkins, Eli Lilly) Potential energy surface for the rearrangement of (R)-litronesib to (S)-litronesib via either a C S or C N bond cleavage pathway. 23

24 Case Study #2 Mechanistic Insight Base, not acid (as expected) catalyzes racemization Relay neighboring group participation required for reaction (position of TWO nitrogens in molecule is critical) C-S cleavage, not C-N Substitution on the aromatic group can enhance or reduce racemization rate Control Acidification of solution formulation Scaffold changes 24

25 Case Study #3: Color Change in API Upon Storage 25

observed Nonhomogeneous, Yellow-orange dots mixed in with orange-red material From

26 Case Study #3: Color Change in API Discoloration of orange-red material, which was a hydrochloride salt of an amine-containing compound Non-homogenous appearance easily observed Nonhomogeneous, Yellow-orange dots mixed in with orange-red material From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14,

10/4/2016 File name/location Case Study #3 - continued Two possibilities based on appearance change Change in particle size Material changes color based on particle size Not likely due to data

27 10/4/2016 File name/location Case Study #3 - continued Two possibilities based on appearance change Change in particle size Material changes color based on particle size Not likely due to data showing no change in particle size after storage at 11 months at 40ºC/75% RH Conversion to free base Color that was observed was lighter than typical free base, but yelloworange color matched color of free base when spread thinly on the packaging Free base HCl Salt 27 Suspected that anti-static amine migrated to liner surface and competed for HCl; served as freebasing agent (pka drug: 9, pka amine: 9.6) From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14, 2012; Also documented in Pharmaceutical Stability Testing to Support Global Markets, Huynh-Ba K, Editor, Springer/AAPS Press, New York (2010)

28 28

29 29 Components of the Bag

30 30 HPLC-UV-MS Analysis

31 10/4/2016 File name/location Example #3: Root cause reactivity with antistatic packaging component IR spectra: Yellow-orange material %Reflectance Blue = HCl salt (API) Red = free base %Reflectance Free base Wavenumbers (cm-1) Wavenumbers (cm-1) Further confirmation using thermal and surface tools From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14,

32 10/4/2016 File name/location Example #3 - Reaction in a closed glass vial -exposed to 40 o C, 75% RH for 2 days with amine (orange) without amine (orange-red) Direct mixing/contact with amine From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14,

33 Case Study #3 Conclusions Mechanistic Insight Basic anti-static agent blooms off packaging liner and deposits onto API Basic conditions causes salt to free base change, resulting in changed color Control Assess need for control based on safety / elegance, quality concerns Change packaging bag? (different liner?) 33

interface following storage at 40 ºC/75% RH for 4 months or 25 ºC/60% RH for 20 months HN O R H 2 N N N H

34 10/4/2016 File name/location Case Study #4: Discoloration of Bulk API Stability samples of white drug substance stored in LDPE liners with laminated foil overwrap exhibited blue spots/lines at the drug-liner interface following storage at 40 ºC/75% RH for 4 months or 25 ºC/60% RH for 20 months HN O R H 2 N N N H 34 From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14, 2012

35 10/4/2016 File name/location Case Study #4: Discoloration of Bulk API Maxplot nm AU nm Discolored Spot 1/7/2005 3:15:42 PM White Drug Substance Sample /7/2005 3:41:09 PM Minutes 35 From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14, 2012

36 10/4/2016 File name/location Case Study #4: Discoloration of Bulk API Peak detected by HPLC analysis is an impurity known to result from reaction of the drug substance with formaldehyde. H 2 N HN O N N H R H O H H 2 N HN O N N H R OH Hydroxymethyl adduct R O NH N NH N N NH 2 NH 2 [O] R O NH H N NH N N NH 2 NH 2 R NH R NH O oxidized dimer brilliant blue in color O Methylene-Linked Dimer 36 From Argentine M, and Jansen PJ, presented at AAPS Workshop on Stress Testing and Degradation, Oct 13-14, 2012

37 Case Study #4 Conclusions Mechanistic Insight Discoloration results from packaging-related formaldehyde reacting with API to form a bluecolored dimeric colored species Previous work in stress testing studies with API and formaldehyde were critical to solving this problem Control Assess need for control based on safety / elegance, quality concerns Change packaging bag (different outer liner?) Care during goose-neck twisting 37

38 Case Study #5 Severe Mass Balance Problem loss of active with no degradation products 38

39 39 pka = 6.3

40 Mass Balance Issue with Tablets but not Capsules 40

41 Stability at 40C/75% RH Capsule Tablet- wet granulated Tablet- direct compression 41

42 42 Stability at 40C/75% RH

43 Volatility of the Free Base vs Maleate Salt 43

44 44 Microenvironmental ph of Formulations

45 Stopping the Salt-to-Base Conversion The phmax was determined to be ~ The microenvironmental ph of the tablet was found to be 4.3, favoring the salt-to-base conversion A stable tablet formulation with shelf-life >3 years was successfully developed by lowering the microenvironemental ph of tablet from 4.3 to <3.0 by adding 2% citric acid to the formulation. 45

46 Case Study #5 Conclusions Mechanistic Insight Mass balance problem was result of volatility Free base was volatile Microenvironmental ph above phmax created conditions favoring salt-to-base conversion Control Stabilize the salt form by lowering microenvironmental ph to phmax or below 46

47 Summary In chemistry, a reaction mechanism is the step by step sequence of elementary reactions by which overall chemical change occurs. For the purpose of this presentation Mechanism= the initiating step and the subsequent cascade Typically, the initiating step is a potential control point. The subsequent cascade is a potential control point, diversion- or stop-point. 47

48 Summary Five Case Studies were presented 1. Artifactual degradation on-column (key variables - column frit, ACN, ammonia, high ph) 2. Unexpected racemization on storage (unprecedented mechanism) 3. API discoloration on storage (packaging antistatic agent induced physical change) 4. A second API discoloration on storage (API reaction with packaging-derived formaldehyde) 5. Significant Mass Balance problem from saltto-free base physical change 48

49 Summary Drug Degradation Mechanisms Can t we just control empirically? Do we really need to develop mechanistic understanding? Is such understanding really a necessary part of Quality-By-Design? 49

50 50 Better Understanding for Better Control Through Chemistry

51 Thank you!!! See baertschiconsulting.com 51

52 52 Back-up Slides

7. Stability indicating analytical method development and validation of Ramipril and Amlodipine in capsule dosage form by HPLC.

7. Stability indicating analytical method development and validation of and in capsule dosage form by HPLC. 7.1 INSTRUMENTS AND MATERIALS USED 7.1.1 INSTRUMENTS 1. Shimadzu LC-2010 CHT with liquid chromatograph