Degradation Mechanism Webinar Baertschi Oct 2107 Seventh Street Development Group.pdf

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1
Drug Degradation Mechanisms: Why
Are They Important?
Steven W. Baertschi, Ph.D.
Baertschi Consulting, LLC
http://baertschiconsulting.com/
Crystal Pharmatech Webinar
October 18, 2017

2
Introduction
I. Drug Degradation Mechanisms
• What is a “degradation mechanism”?
• Do we really need to develop “mechanistic understanding”?
• Can’t we just control empirically?
II. Seven 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)
6. Unexpected photo-instability
7. Severe oxidative instability
III. Conclusions

3
Better Understanding
for Better Control
…Through Chemistry

4
What I Intend to Convey:
Take Home Message
• We want to develop an understanding of
“mechanism”, chemical or physical, such that we
can develop control strategies that are:
✓Practical (implementable)
✓Robust
✓Effective
✓Timely
✓Economical

5
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.
• It often involves kinetics (e.g., rate determining step)
• Order of reaction
• Movement of electrons, protons, atoms
• Balanced equation
• Etc.
• 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”.

7
Ea Ea, uncat.
Ea, cat.
Metastable state
Deg product 2
Deg product 1
Deg product 3
Deg product 4
Initiating Step
Resulting Cascade

8
Case Study #1: Unexpected New
Peaks in Chromatogram
Unexpected
New Peaks

9
Optimum Conditions for Litronesib based
on Column Screening Strategy
• 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.
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)
% B (Acetontrile)
0 75 25
1 75 25
23 20 80
25 20 80
25.1 20 80
28 20 80
Autosampler temperature 5ºC
Detection wavelength 290 nm
Column temperature 30ºC
Injection volume 10 μL

10
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

11
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% NH4OH
Mobile Phase B- Acetonitrile
Acc. Mass MS =
Parent + N,O - H
How do we
control?

12
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 NaNO2 and pH to 1 with HCl

13
So how is Litronesib being N-
nitrosylated on-column??
• Where does the N=O come from?
• Acetonitrile? -- No (pardon the unavoidable pun)
• Ammonia? – Yes!
• 15N-labelling experiment showed that the nitrogen
came from ammonia.
• Griess test (for nitrite) showed positive formation of
NOX eluting from the column
Griess test reaction
Colored
species

14
On-column reaction increases with
column use…
Relative total nitrosamine peak area percents (UV at 290 nm) for
consecutive injections on the column

15
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)

16
min
5 10 15 20 25 30
mAU
0
5
10
15
20
*DAD1 A, Sig=290,16 Ref=400,10 (D:DATAEG5 2012DM JAN13_1DM_JAN_13_2 2012-01-13 13-58-58002-0301.D)
*DAD1 A, Sig=290,16 Ref=400,10 (D:DATAEG5 2012DM JAN13_1DM_JAN_13_2 2012-01-13 13-58-58002-0302.D)
*DAD1 A, Sig=290,16 Ref=400,10 (D:DATAEG5 2012DM JAN13DM_JAN_13_1 2012-01-13 08-43-22002-0301.D)
Before and After EDTA added to Mobile PhaseA
Standard Mobile Phase A
Standard Mobile Phase Aw/EDTA
Standard Mobile Phase Aw/EDTA
X-Bridge C18 Separations with 50 uM EDTA
added to the mobile Phase-LC UV data
Waters X-Bridge C18, 4.6 x 75 mm, 2.5 um
Mobile Phase A- 0.05% NH4OH + 50 uM EDTA Mobile Phase B- Acetonitrile
EDTA reduces total nitrosamines
peak area by ~ 10x

17
Current Hypothesis for Mechanism of
Nitrosamine formation
Source of metal ion M+ is likely in all stainless steel
inlet frits used in column hardware
2Carcinogenesis vol.18 no.9 pp.1851–1854, 1997

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• Initiating Step
• HPLC analysis under certain conditions  analytical artifact
• 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
So how is Litronesib being N-nitrosylated
on-column??
Ea, cat. Ea, uncat.
NH3
NOx

19
Case study #2: Unexpected
racemization
Ea, cat. Ea, uncat.
Protonated
Deprotonated
Racemized

20
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)

21
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.

22
No mechanistic insight…scaffold
risks?
• Since no mechanism could be developed, insight
was sought to enable future scaffold improvements
• Experiments designed to probe mechanism

23
Proposed Mechanism Based on
Experimental Data

24
Probing the Proposed Mechanism:
Experiments with Two Analogs

25
Final Question: C-N or C-S cleavage?

26
Computational studies clearly
implicate C-S cleavage
Potential
energy surface
for the
rearrangement
of (R)-litronesib
to (S)-litronesib
via either a C–
S or C–N bond
cleavage
pathway.
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)

27
Case Study #2
• Initiating Step
• Base-catalyzed neighboring group
participation
• 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

28
Case Study #3: Color Change in API
Upon Storage
Ea, cat. Ea, uncat.

29
9
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
Non-
homogeneous,
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, 2012

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File name/location
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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 yellow-
orange color matched color of free base when spread thinly on the
packaging
• Suspected that anti-static amine migrated to liner surface and
competed for HCl; served as freebasing agent (pKa drug: ≤9, pKa
amine: 9.6)
HCl Salt
Free base
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)

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File name/location
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Example #3: Root cause – reactivity
with antistatic packaging component
98.1
98.2
98.3
98.4
98.5
98.6
98.7
98.8
98.9
99.0
99.1
99.2
99.3
99.4
99.5
99.6
99.7
99.8
99.9
%Reflectance
500
1000
1500
2000
2500
3000
3500
4000
Wavenumbers (cm-1)
98.1
98.2
98.3
98.4
98.5
98.6
98.7
98.8
98.9
99.0
99.1
99.2
99.3
99.4
99.5
99.6
99.7
99.8
99.9
%Reflectance
500
1000
1500
2000
2500
3000
3500
4000
Wavenumbers (cm-1)
99.0
99.5
100.0
100.5
101.0
101.5
102.0
102.5
103.0
103.5
104.0
%Reflectance
500
1000
1500
2000
2500
3000
3500
4000
Wavenumbers (cm-1)
99.0
99.5
100.0
100.5
101.0
101.5
102.0
102.5
103.0
103.5
104.0
%Reflectance
500
1000
1500
2000
2500
3000
3500
4000
Wavenumbers (cm-1)
Blue = HCl salt (API)
Red = free base
Yellow-orange
material
Free base
IR spectra:
Further confirmation using thermal and surface tools

34
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File name/location
4
Example #3 - Reaction in a closed glass
vial -exposed to 40oC, 75% RH for 2 days
with amine
(orange)
without
amine
(orange-red)
Direct mixing/contact with amine

35
Case Study #3 Conclusions
• Initiating Step
• Packaging interaction
• 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?)
Ea, cat. Ea, uncat.
salt
Free base

36
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File name/location
6
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
R
N
H
N
HN
O
H2N

37
Components of the “Bag”

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File name/location
8
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
1/7/20053:15:42PM
1/7/20053:41:09PM
DiscoloredSpot
WhiteDrugSubstanceSample
Maxplot380-590nm
nm
300.00 400.00 500.00 600.00

39
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File name/location
9
• Peak detected by HPLC analysis is an impurity known to
result from reaction of the drug substance with formaldehyde.
R
N
H
N
HN
O
H2N
H H
O
R
N
H
N
HN
O
H2N
OH
R
NH
N
NH
O
NH2
R
H
N N
NH
O
NH2
R
NH
N
NH
O
NH2
R
N N
NH
O
NH2
[O]
Hydroxymethyl adduct
Methylene-Linked Dimer
oxidized dimer
brilliant blue in color

40
• Initiating Step
• Formaldehyde from package liner migrating through
cracks induced by goose-necking
• Discoloration results from packaging-related
formaldehyde reacting with API to form, upon
oxidation, a blue-colored dimeric 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

41
• Case Study #5
• Severe Mass Balance Problem – loss
of active with no degradation products
Ea, cat. Ea, uncat.

43
Mass Balance Issue with Tablets but
not Capsules

44
Stability at 40C/75% RH
Capsule
Tablet- wet
granulated
Tablet- direct
compression

45
Stability at 40C/75% RH
Loss of active,
loss of total mass
detected

46
Volatility of the Free Base vs Maleate Salt
pKa = 6.3

47
Microenvironmental pH of Formulations

48
Disproportionation (dissociation) of Salts







 5
.
0
0
max log
Ksp
S
pK
pH a
ΔG ≈ - RT ln(S salt/S freebase)
Region 1 Region 2
Figure 2. Solubility diagram of salts of a weak base having an intrinsic solubility of 1 mg/mL, and pKa of 5.0 with salt
forms a (hydrochloride), b (sulfate) and c (tosylate), having solubility’s of 200, 50 and 10 mg/mL, respectively.
Stephenson GA et al., Physical Stability of Salts of Weak Bases
in the Solid State, J. Pharm. Sci. 100:5 (2011) 1607-1617
HCl
Sulfate
Tosylate

49
Stopping the Salt-to-Base Conversion
• The pHmax was determined to be ~3.3-3.6
• 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
microenvironmental pH of tablet from 4.3 to <3.0 by
adding 2% citric acid to the formulation.

50
• Initiating Step
• Salt to free base conversion
• Mass balance problem was result of volatility of
free base
• Microenvironmental pH above pHmax created
conditions favoring salt-to-base conversion
• Control
• Stabilize the salt form by lowering
microenvironmental pH to pHmax or below
Ea, cat.
salt
Free base

52
• Case Study #6
• Unexpected / Unpredicted
Photoinstability
Ea
Epinephrine

Epinephrine
• pKa1 8.69; pKa2 9.091
• Easily oxidized
• Stabilized by sulfites
Szulczewski DH, Hong W. Epinephrine,
in Analytical Profiles of Drug
Substances. Academic press, Inc. 193-
229, 1978
No UV absorption
above 300 nm!!

54
Unexpected Photo-Instability
HO
HO
NHCH3
OH
O
–
O N+
OH
CH3
O
–
O N+
OH
CH3
SO3
–
Na+
O
–
O N+
OH
CH3
SO3
–
Na+
Epinephrine Adrenochrome
Adrenochrome
sulfonate
*
hv
350 nm
O2
1
O2 O2
HSO3
–
Na+
–
•A well defined example: bisulfite in diluents leading to photoinstability of
catecholamines
J. Brustugun, H. H. Tonnesen, W. Klem, I. Kjonniksen PDA Journal of
Pharmaceutical Science and Technology 2000; 54: 136-143.
Photosensitizer
54

55
Case Study #6
Conclusions
• Initiating Step
• Bisulfite reaction with oxidative deg product
• Bisulfite adduct is a photosensitizer (350 nm)
• Singlet oxygen (and other ROS) are then formed
• Epinephrine is not itself photosensitive
• Control
• Eliminate the antioxidant bisulfite
Ea
Oxidation product
Epinephrine
Reactive Oxygen
Species
Bisulfite adduct
350 nm

56
• Case Study #7
• Significant oxidative instability
Metastable state

• Pemetrexed (Alimta®), a folate analog metabolic
inhibitor
• Liquid formulation desired
• Significant oxidative instability observed
• Forced degradation studies revealed the degradation
products and mechanistic insight 57

60
• Initiating Step
• Tautomerization to phenol / deprotonation
• Phenol tautomer highly susceptible to
base, metal, photo-catalyzed oxidation
• Initial oxidative step leads to cascade of reactions /
multiple products
• Control
• Freeze-dried formulation vs liquid
• Inerting (nitrogen) / antioxidants
• pH lowering
• Protect from light
Tautomer
(Metastable state)
Deprotonated
form
Multiple
products

61
Summary
➢ Seven Case Studies were presented
1. Artifactual degradation on-column (key variables - column frit,
ACN, ammonia, high pH)
2. Unexpected racemization on storage (unprecedented
mechanism, pH control)
3. API discoloration on storage (packaging anti-static agent
induced physical change)
4. A second API discoloration on storage (API reaction with
packaging-derived formaldehyde)
5. Significant Mass Balance problem – from salt-to-free base
physical change
6. Drug with no UV absorbance exhibits photoinstability – from
rxn of oxidative deg product with bisulfite and light!
7. Oxidative instability – tautomerization and deprotonation
leads to instability

62
Summary
➢Drug Degradation Mechanisms
• What is a “degradation mechanism”?
• Do we really need to develop “mechanistic
understanding”?
• Can’t we just control empirically?

63
Summary
Metastable state
Deg product 2
Deg product 1
Deg product 3
Deg product 4
Initiating Step
Resulting Cascade
• 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”.

64
What I Intend to Convey:
Most Important Take Home Message
• We want to develop an understanding of
“mechanism”, chemical or physical, such that we
can develop control strategies that are:
✓Effective
✓Robust
✓Practical (implementable)
✓Timely
✓Economical

65
Better Understanding for
Better Control
…Through Chemistry

66
Thank you!!!
See baertschiconsulting.com

Degradation Mechanism Webinar Baertschi Oct 2107 Seventh Street Development Group.pdf

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