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Analytical method development for the determination of drug

degradation: case studies.Claudio Brunelli

Pfizer Analytical Research and Development

Pfizer Confidential

Amsterdam 14-15Oct2019│ 1

Analytical requirement

- The important requisite for stability studies and forced

degradation study is to submit the stressed samples to

dedicated reliable analytical methods that:

ensure all degradants are captured

ensure all degradants are identified

provide substrate for method development and optimization

- We recommend to full degradation protocol

- We recommend to screen the samples generated at each

degradation condition with a pool of orthogonal analytical

methods

SOS – Amsterdam 14-15Oct2019 │ 2

Case Study 1: method development

- How to develop a method when sample and impurities are not

available?

- What should the method developed for?

- Use the sample set provided by ZENETH

A Virtual Key Predictive Sample Set (vKPSS) could be

generated

The method will be developed for the structures generatedSOS – Amsterdam 14-15Oct2019 │ 3

Case Study 1: method development


- Generate a virtual chromatogram:

- The retention of all the compounds related to the

synthetic route up to step previous the API were

available.

- Retention data on the most similar compounds it was

possible to predict the retention of the expected

degradation products

- Chromatpgraphy simulation software are becoming

available: LC-simulator (ACD-labs) in prediction mode


Case Study 1: virtual method development

Structure

PRI 1

PRI 2

PRI 3

API

Structure 1

Structure 2

Structure 3

Structure 4

Structure 5

Structure 6

Structure 7

PRI 4

The model was built on

the retention data of

the Step4 impurities.

The Zeneth expected

degradants were

predicted and

modelled.

The chromatogram

was simulated

excluding the Step 4

imps and including only

the API degradants.



Build virtual temperature / gradient modelling retention

Retention data

The retention times of compounds related to API (precursors and starting

materials) were captured in six temperature and gradient time conditions:

T = 10, 25 and 40°C

GT = 25 and 60 min

Simulated Temperature / gradient

The retention of vKPSS was predicted in six conditions using LC-Simulator

And the modelled in the usual way



VERIFICATION

The optimized method

conditions were applied on a

real sample

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14.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.3814.38 16.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.1716.17

16.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6516.6518.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.4318.43

20.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.0020.00

21.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.3721.37

23.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.0223.02

21.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.0421.04

23.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1023.1022.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9722.9724.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.9824.98

25.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.8125.81

Oxid

AP

I

Ste

p 4

Oxid

Cis

-AP

I /A

PI

Ste

p 4

Cis

-AP

I

R1

N

NH

O

R2

R3

R1

R3

N

NOH

R2

1 2



Oxid

AP

I

Ste

p 4

Oxid S

tep

4

Cis

-AP

I

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7.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.937.93

8.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.818.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.398.39

10.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.3210.32

10.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.5910.59

14.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5514.5516.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.5316.53

17.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.0117.01

17.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.9917.99

20.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.9720.97

23.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.2223.22

21.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7921.7923.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.3823.38

21.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.3921.39

23.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.2523.25

25.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.0425.04

25.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.8625.86

19.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.9219.92

VERIFICATION

Tautomer of Oxid Deg was

used with agreement on

retention: what is the real form

of the impurity?

WARNING: Limitation with

regards to stereoselectivity


Cis

-AP

I /A

PI


• Purity and stability methods were required for the analysis of a drug

product in development.

• Minimal knowledge was available and formulations options were still

under evaluation

• A series of samples were analysed with a set of generic

chromatographic conditions (COMET) in order to determine all the

key components

• COMET analysis (Comprehensive Orthogonal Method Evaluation

Technology) consists of a combination of conditions set to maximize the

selectivity space so that all constituents can be detected in at least one

method.

1. Three pH: acidic, neutral and basic

2. Four stationary phases

3. Two organic solvents

Case Study 2: On column degradation

SOS – Amsterdam 14-15Oct2019 │

10

- Question 1: Do we see everything? Did we investigate the

samples with thorough orthogonality?

- Predictive software and chemistry check didn’t anticipate

the possibility of isomers, non ionizable or non

chromophoric impurities, or highly polar or highly

hydrophobic substances or anything not suitable for

reversed phase LC.



• With any stationary phase and organic solvent used for method

development screening analyses, the number of peaks differed

depending on the mobile phase pH used. In fact, two different scenarios

were presented:

• At acidic pH (0.1% formic acid, pH = 2.6) one key potential

degradation product was not detected (Scenario 1)

• Only at basic pH (0.1% ammonium hydroxide, pH = 10.8) three high

mw peaks could detected (Scenario 2)



5.67

5

5.85

2

6.73

6

7.03

3

7.16

5

7.27

4

7.38

7

9.04

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Minutes

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API (80.7%)

FA1 (7.4%)

FA2 (10.7 %)

API (94.5%)

FA1 (5.5%)

pH = 6.8

pH = 2.6

The formaldehyde derived degradation impurities (FA1 and FA2) seem to

degrade when acidic pH was used. This was evident on all the stationary

phases screened

It is not a co-elution as confirmed by MS



- Question 2: Do we see too much? Are all the observed

peaks real impurities to be determined or is any of these

artificially generated during the chromatography?

- Co-elution was excluded by mass spectrometry. Analyte could be

reacting or degrade with stationary or mobile phase.



• Three peaks only appeared when a basic mobile phase was used.

Their level and presence were not consistent across or different

condition. Such lack of consistency raised the hypothesis that

these were formed on column

mw 2 x mw – 2 + 2 x mw – 4

• MS suggests structures related to API, azo and hydrazo dimers.

• It was necessary to confirm or reject this possibility to ensure that

no real impurities were missed

R1

NH

R2

R1

NH

R2

R1

N

R2

R1

N

R2

+

R1

NH2

R2

4 X



Correct assignment of the observed peaks is of crucial

importance:

• Conditions that can generate artefacts could make the method

unusable.

• Conditions that can degrade critical impurity could compromise the

stability program and eventually compromise the quality and safety of

the final product.

• It was necessary to establish which peaks were artificially

generated on column and differentiate them from the sensitive

ones requiring accurate and precise quantification



• Two dimensional LC combines two different separation modes or

methods in the first and second dimension, with selectivity as

orthogonal as possible:

• Heart-cut: is suitable for the re-analysis of defined number of peaks in

order to separate peaks otherwise co-eluting in one dimension taking

advantage of the orthogonal selectivity. Typical setups are:

• acidic - basic reversed phase (RP)

• RP-HILIC

• Normal phase (NP) – RP

• SFC – RP

• NP/RP – chiral…

• Comprehensive mode: each section of the chromatogram in the first

dimension is scrutinized in the second dimension: it increases the peak

capacity several times and becomes and it is best option for the analysis of

very complex matrix (e.g. proteomics, biomarkers, natural extracts etc…)



• The first dimension was set up with basic mobile phase reproduce

/ separate the suspect peaks.

• Experiment 2: the second dimension was set up with using same

stationary phase (BEH C18), with same, basic, mobile fractions of

main band were re-analysed in order to regenerate the artefacts.

2D-LC settings:

Columns: on both first and second dimension a BEH C18 was used, 100

mm x 2.1 mm, 1.7 μm ps. A new column was installed in the second

dimension.

Mobile phase:

1D: 0.1 %NH4OH, pH = 10.8

2D: Experiment2: 0.1 %NH4OH, pH = 10.8.



The main band was sampled with different size and time slices. Each cut

was re-injected in the second dimension which used basic mobile phase

(that is: exactly same conditions as first dimension).

Heart-cut details:Fraction 1D Time Sampling time

(min) (min)

Fr.1) 8.10 0.05

Fr.2) 8.27 0.01

Fr.3) 8.30 0.01

Fr.4) 8.35 0.05

Fr.5) 8.43 0.05

Fr.6) 8.77 0.05



Fraction 2

Fraction 3

Fraction 4

Fraction 5



This second experiment confirmed beyond any doubt that the mentioned peaks are

artefact and that they formed only during the chromatographic process on a basic

mobile phase.

Overlay of fractions 3, 4 and 5 analysed in the second dimension.



mw mw + 30 + 2 x mw +12

Formaldehyde is an impurity present in several excipients, such as lactose.

Its reactivity is responsible of several degradation derivatives present in

several drug products.

Two are the main products derived by the reaction of the API with

formaldehyde.

R1

NH2

R2

R1

R2

NH

OHCH2 O

R1

R2

NH

R1R2

NH

+

Case Study 2: more on column degradation


• The neutral pH offered the highest robustness and separation for

most of the KPSS and the method development was continued

using neutral.

• Impurities derived from degradation in excess of formaldehyde were

degrading when acidic pH mobile phase was used.

• The separation between the main band and the one of the main

formaldehyde derived impurities was more than baseline nevertheless

the peculiar shape led to suspect that an on column degradation was

occurring.

• This was investigated and confirmed by use of 2D-LC.



min12 12.25 12.5 12.75 13 13.25 13.5 13.75

mAU

-9

-8

-7

-6

-5

-4

DAD1 A, Sig=225,4 Ref=off (brunec01\P...de degradants\CB_06May15_TM100005211_1D 2015-05-06 11-56-39\FA.D)

Are

a: 5

60.107

13.1

76

When analysed with neutral conditions, the particular shape leads to suspect that a

degradation, conversion or some sort of reactivity of the two peaks occurs.

min12 14 16 18 20 22 24 26 28

mAU

-20

0

20

40

60

80

100

DAD1 A, Sig=225,4 Ref=off (brunec01\P...de degradants\CB_06May15_TM100005211_1D 2015-05-06 11-56-39\FA.D)

Are

a: 4

8.58

37

12.7

84

Are

a: 5

09.64

13.1

76

Are

a: 3

73.228

26.0

20

AP

I F

A1

FA

2

AP

I FA

1



The same conditions were applied on both dimensions, including stationary

phase, mobile phase an instrumental parameters in order to determine if the

pure peak converts into the API as suspected.

Columns: both first and second dimension were equipped with a BEH C18 was used,

100 mm x 2.1 mm, 1.7 μm ps.

Mobile phase: A: 10 mM ammonium acetate, pH=6.8, B: Acetonitrile

Settings (both dimensions except injection volume):

Temperatures: 55°C; Flow Rate: 0.4 mL/min; Injection volume (D1):10 μL

Gradient program: Time (min) % Organic

0 10

4 10

30 50

35 95

35.5 10

38 10



• FA1 peak was collected and re-injected in order to observe its behaviour.

• The fraction between the two peaks was also collected in order to

determine its composition.

Heart-cut details:

Fraction 1D Time Sampling time

(min) (min)

Fr.1) 12.64 0.01

Fr.2) 12.80 0.05

Fr.3) 13.07 0.05

Fr.4) 25.88 0.05



The same time fractions were also collected from a control sample, in order

to exclude that the presence of any peak could be due to not syncd fraction

collection. Below is the overlay of the chromatograms comparing the control

(blue) and the degraded sample (red) and zoom of the time of interest.

min12 12.5 13 13.5 14

mAU

-10

-8

-6

-4

-2

0

2

4

6

DAD1 A, Sig=225,4 Ref=off (C:\CHEM32\...ANTS\CB_15MAY15_TM100005211_2D 2015-05-15 10-38-35\API Control.D)

13.0

47

DAD1 A, Sig=225,4 Ref=off (C:\CHEM32\...DE DEGRADANTS\CB_15MAY15_TM100005211_2D 2015-05-15 10-38-35\FA.D)

12.6

98

13.0

85

min10 12 14 16 18 20 22 24 26 28

mAU

-25

-20

-15

-10

-5

0

5

10

15

DAD1 A, Sig=225,4 Ref=off (C:\CHEM32\...ANTS\CB_15MAY15_TM100005211_2D 2015-05-15 10-38-35\API Control.D)

13.0

47

DAD1 A, Sig=225,4 Ref=off (C:\CHEM32\...DE DEGRADANTS\CB_15MAY15_TM100005211_2D 2015-05-15 10-38-35\FA.D)

12.6

98

13.0

85

25.8

99

AP

IFA

1



Fraction 1:

The reanalysis of the fraction 1 shows that a significant amount of FA1 is

reverted back to API during the chromatography up to 25%.

Peak Area Area %

FA1 352.15 75.5

API 114.117 24.5

API

FA1



The analysis of the fraction collected between the two peaks also reveals

the presence of a mix of both FA1 and API, of course in lower amount. The

ratio is shifted toward API.

Fraction 2:

APIFA1



In order to demonstrate that the peaks observed in the re-injected fractions

on second dimension are not due to fronting of the main band, they have

compared with the same fractions collected from a control sample and, as it

can be observed from the chromatogram above, the fraction is clean

Fraction 3:



In fraction 4 also reveals the presence of the API at a level of 0.4% (not a

carryover as confirmed from the control analysis).

The large peak (2.3%) before the API is peak of unknown origin but is not

observed in the first dimension.

Fraction 4:

FA2

API



Case Study 3: method risk assessment

- The project chromatographic method was applied to drug

product in stability. It was generated after forced degradation of

the API

- The method revealed a series of late eluters

AP

I

Dim

er

Trim

er

Tetr

am

er

95% organic

- Control

- Degraded

*



- The presence of very apolar components raises the question of

the suitability of the current chromatographic method: the

elution at high organic has to be extended.

- A second question regards the sample preparation: it needs to

ensure that all possible apolar degradants are in fact extracted

- In addition to the known dimer, the presence of a trimer and

tetramer was revealed (Mass Spec).



- The solubility of dimer, trimer and tetramer was determined in-

silico (markers not available)

- The solubility decreases nearly 1000 times with every addition of

a monomer. This prompts to review the appropriate sample

preparation to ensure the dissolution the oligomers as well as

ever other potential deg with even higher LogD

Deg LogS (pH = 5.00) LogS (pH = 7.00) LogS (pH = 9.00) LogD (pH = 5.00) LogD (pH = 7.00) LogD (pH = 9.00)

API -4.33 -4.08 -2.39 1.91 1.66 0.33

Dimer -7.11 -7.72 -6.18 3.97 4.75 3.52

Trimer -10 -11.46 -10.08 6.04 7.71 6.59

Tetramer -12.78 -15.09 -13.86 8.38 10.89 9.89



- Although the dimer is the main degradant, and therefore the first

to breach the spec, although newer formulations will likely have

higher loading (expecting therefore lower overall degradation),

we need to ensure the accurate recovery of all degs through

appropriate sample recovery


- The development of a stability indicating method is one of the highest

priorities in the activities of pharmaceutical laboratories during drug

development and is a re-iterative

- The availability of key degradation components is critical, however the

accurate determination of the degradation products under different stress

conditions is critical and heavily rely on a suitable analytical method.

- The combination of degradation profile prediction with chromatographic

structure-retention modelling and physicochemical properties computation

facilitate method development.

- On-column reactivity can compromise the suitability of a chromatographic

method

Conclusions


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