Natural Stable-Isotopic Records of Pharmaceutical Compounds:
Anticounterfeiting and Process Patent Protection
John P. Jasper1, L. E. Weaner2,
R. C. Lyon3, and J. M. Hayes4
1Nature’s
Fingerprint® / MIT LLC, Niantic, CT 06357 USA;
2Johnson & Johnson Pharmaceutical R and D,
Spring House, PA 19477, USA;
3Division of Product Quality Research, CDER, FDA,
Silver Spring, MD 20993 USA;
4Woods Hole Oceanographic Institution,
Woods Hole, MA 02543 USA.
Product
Authentication
Quality Control
Process
Authentication
Batch Identification
Process Analytical
Chemistry (PAC)
•Qualification of Raw Materials
•Anticounterfeiting
•Uniformity of Manufacturing
•Patent Protection
Product and Process Authentication
-26
-28
-30
-32
-34
-36
-38
-40
-42
-44
Site 1
Site 2
C a r b o n ( o /o o )
Reactant
Reactant
-
1
-5
-5
Product
-1
Product
0
1
2
3
4
5
6
Nitrogen (o/oo)
7
8
9
10
Outline
1. Product Authentication
a. Qualification of Raw Materials
b. Batch Auditing
2. Process Authentication
a. Process Understanding
b. Process Patent Protection (PPP)
3. PPP Summary Presentation
Product Authentication by Isotopes
[Jasper, 2004, Tablets & Capsules 2(3):37-42]
Isotopic Provenance
= f(reactants, synthetic process)
Isotopic Composition ()
Key Mechanism: Isotope Fractionation
Reactants
Reaction
Product

A+B
(A-B)*
Time
C
Pharmaceutical Materials
1. Active Pharmaceutical Ingredients (APIs)
2. Excipients
3. Drug Products (APIs + Excipients)
Elemental Analyzer/Mass Spectrometer (EAMS)
Reference Gas
Injector
Elemental Analyzer
CO2 He N2
02
He
Mass
Spectrometer
Flash
Combustion
T=1800°C
-H2O
Time
IRMS
GC Column
Oxidation Reduction
Part 1. Product Authentication*
•Batch Processes
•Anticounterfeiting
*US Patent Issued to MIT LLC on Jan. 29, 2008.
1A. OTC Analgesic Drug Products:
Batch-to-Batch Variation
•One Drug Product
•One Manufacturer
•Thirty Batches
18
 O Records by Batch Order: Acetaminophen
20
16
12
8
29
27
25
23
21
19
17
15
13
11
9
7
5
3
4
1
18
 O (‰ v s V S M O W )
24
Batch Order (By Expiration Date)
[Pharm. Tech. 2004, 28(8):60-67]
20
1.B.1. APIs:
Differentiating Manufacturers:
•One API
•Six Manufacturers
•Four Countries
13
 C (‰ vs V P D B )
Naproxen: 13C vs. 18O
-24
India,
Mfr B
-26
-28
Italy,
Mfr C
Ireland,
Mfr E
-30
India,
Mfr A
Italy,
Mfr D
-32
USA, Mfr F
-34
-5
0
5
10
15
20
18
 O (‰ vs VSMOW)
(Wokovich et al., 2004, JPBA 38:781-784)
1.B.2 APIs:
Differentiating Manufacturers:
•One API
•Three Manufacturers
•Three Countries
Folic Acid From 3 Pharmaceutical Companies:
13
15
 C vs.  N
-23.5
-24.0
Switzerland
13
 C (‰ vs Air)
-24.5
-25.0
-25.5
-26.0
India
-26.5
-27.0
Japan
-27.5
-28.0
-18
-15
-12
-9
-6
15
 N (‰ vs Air)
-3
0
3
1.C. Batch Auditing / Batch Smearing
•Batches Shifted
•Batches Mixed
Part 2. Process Understanding*:
•PAC
•Process Patent Protection
*US Patent Pending to MIT LLC.
“Characterizing Synthetic Pathways
By Stable-Isotopic Measurements”
(Jasper, Weaner, & Hayes,
Pharm. Tech., March, 2007)
Complete Record of Stable-Isotopic Synthesis
+
A: -30.0‰ , 0.25, -10‰
B: -15‰ , 0.05, -30‰
C: -25.5‰ , [-24.4‰ ]
D: -10‰ , 0.05, -30‰
E: -20.4‰ , [-18.2‰ ]
+
C: -25.5, 0.50, -30‰
+
E: -20.4‰ , 0.10, -15‰
F: -15‰ , 0.30, -5‰
G : -19.1‰ . [-17.2‰ ]
C8
+
G : -19.1‰ , 0.20, -15‰
H: -30.0‰ , 0.10, -15‰
I: -22.0‰ , [-20.2]
13
 C (‰ v s V P D B )
Isotopic Records of Products
With and Without Isotopic Fractionation
-17
-18
-19
-20
-21
-22
-23
-24
-25
-26
G
E
I
C
0
1
2
3
Product Number (n)
4
Isotopic Differences Caused by Fractionation
 P*  P (‰ )
2.5
2.0
E
1.5
1.0
G
I
3
4
C
0.5
0.0
0
1
2
Product Number (n)
5
Carbon-Isotopic Fingerprints of Three Synthetic Pathways
-25
Route B
-29
Starting
Material
-31
Route A
Intermediate
Product
-33
13
C (‰ vs. VPDB)
-27
-35
-37
Authentic
-39
0
Fig. 3a
1
2
3
Compound (n)
4
5
Process Patent Protection
Schematized Patent Infringement Case
-25
Products
Processes
13C (‰)
-27
Plaintiff
-29
-31
Defendant
-33
-35
0
1
2
3
4
5
6
7
8
9
10
Compound Number (n)
© Evans Analytical Group 2011
25
The Isotopic Record of Pharmaceutical Synthesis
-------------------Precursors ---------------
Starting Materials
Synthetic Intermediates
Product
A + B  C
C + D  E
E + F 
Int./Products C
Precursors
A , B
Fraxn Rmng. fA, fB
Isotopic Frxtn. C
E
C, D
fC, fD
E
G
G
E, F
fE, fF
F
Three Levels of Understanding
1. Isotope Mass Balance.
2. Isotope Mass Balance with 1 Excess Component.
3. Isotope Mass Balance with 2 Excess Components.
The Basic Format:
Isotope Mass Balance
Excess
Components
0:
mAA + mBB = mCC
1:
mA(A - A) + mBB = mCC
2:
mA(A - A) + mB(B - B) = mCC
Overall…
•Isotopic compositions are controlled by
reactants and processes.
•Variations from mass balance are due to
isotope effects of specific processes.
Part 3.
Summary of Process Fingerprinting
via Stable Isotopes
Product Fingerprint for Authentic API:
Manufacturing Site 1
-26
-28
C a r b o n ( o /o o )
-30
-32
-34
-36
Authentic API, Site 1
-38
-40
-42
-44
0
Fig. 1
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Starting Material and Authentic API:
Overall Reaction (5 Steps Not Shown Here)
-26
Starting Material,
Source 1
-28
C a r b o n ( o /o o )
-30
-32
-34
-36
Authentic API,
Site 1
-38
-40
-42
-44
0
Fig. 2
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Process Fingerprint: Isotopic PedigreeR for Authentic API
-26
Starting Material, Source 1
-28
C arbon (o/oo)
-30
-32
-5
-1
-34
-36
Authentic API, Site 1
-38
Process Fingerprint = Vector (-5 C, -1 N)
-40
-42
-44
0
Fig. 3
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Product Fingerprint for Authentic API:
Manufacturing Site 2
-26
-28
C a r b o n (o /o o )
-30
-32
-34
-36
-38
Authentic API, Site 2
-40
-42
-44
0
Fig. 4
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Starting Material and Authentic Product:
Manufacturing Site 2
-26
-28
Starting Material,
Source 2
C a rb o n (o /o o )
-30
-32
-34
-36
-38
Authentic API,
Site 2
-40
-42
-44
0
Fig. 5
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
R
Process Fingerprint: Same Isotopic Pedigree
Generated at Two Sites
-26
Starting Material, Source 1
-28
Starting Material, Source 2
C a r b o n (o /o o )
-30
-32
-34
-36
Authentic API, Site 1
-38
Authentic API, Site 2
-40
-42
-44
0
Fig. 6
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Alternative Manufacturing Process:
Non-Infringing
-26
-28
Fingerprint
C N .
Generic Process +2 -3
Authentic Process -5 -1
C a r b o n (o /o o )
-30
-32
-34
-36
Generic Product
-38
-40
-42
Starting Materials
-44
0
Fig. 7
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Alternative Manufacturing Process:
Non-Infringing
-26
-28
C ar b o n (o /o o )
-30
-32
-34
Process Fingerprint = Vector (+2 C, -3 N)
-36
Generic Product
-38
-3
+2
-40
-42
Starting Material
-44
0
Fig. 8
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Alternative Manufacturing Process:
Non-Infringing
-26
Authentic 1
-28
Authentic 2
C a r b o n (o /o o )
-30
-32
-5
-34
-1
-36
-38
-40
Generic
-42
-44
0
Fig. 9
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Generic Sample Likely Infringing Authentic Process
-26
-28
C a r b o n ( o /o o )
-30
-32
-34
-36
Generic
product
-38
-40
-42
-44
0
Fig. 10
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Generic Sample Likely Infringing Authentic Process
-26
Reconstructed
Generic Reactant
-28
C a r b o n ( o /o o )
-30
-32
-34
-36
Generic
Product:
Likely Infringing
-38
-40
-42
-44
0
Fig. 11
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Generic Sample Infringing Authentic Process
-26
Authentic
-28
Authentic
C a r b o n ( o /o o )
-30
-32
-5
-34
-36
-1
-38
-40
Generic sample
-42
-44
0
Fig. 12
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Generic Sample Infringing Authentic Process
-26
Authentic
-28
Authentic
C a r b o n ( o /o o )
-30
Infringing
Generic
-32
-5
-34
-36
-5
-38
-40
-1
-1
Generic sample infringing.
-42
-44
0
Fig. 13
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Generic Sample Infringing Authentic Process
-26
Authentic
-28
Authentic
C a r b o n ( o /o o )
-30
Third Source of
Starting Material
-32
-5
-34
-36
-1
-38
-40
Generic sample infringing.
-42
-44
0
Fig. 14
1
2
3
4
5
Nitrogen (o/oo)
6
7
8
9
10
Summary:
Major Applications for Pharmaceutical Isotope Analysis
1. Product Authentication
a. Qualification of Raw Materials
b. Batch Auditing
2. Process Authentication
a. Process Understanding
b. Process Patent Protection (PPP)
Isotopic Processes and Products
Precursors
R
Processes
A
Products
P
•Product Identification
•Process Consistency and Patent Protection
Patents Granted and Pending
•Product Authentication (Anticounterfeiting)
•Process Authentication (Patent Protection, PAC)
FDA Disclaimer
The views presented here
do not necessarily reflect those of the
Food and Drug Administration.
www.NaturesFingerprint.com
Part 3.
Summary of Process Fingerprinting
via Stable Isotopes
1.D Monitoring Proportions
of API and Excipient
•One API
•One Excipient
•Continuum of Mixtures
Carbon-Isotopic Composition Reflects
the Percentage Compositions of API and Excipient
100
C o m p o sitio n (% )
90
80
80% API
70
60
%API
50
%Excip
ient
40
30
20
20% Excipient
10
0
-27
100% API
-24
-21
-18
 C (o/oo vs VPDB)
13
-15
-12
100% Excipient
1.E. Monitoring Fractional Composition
of a Four-Step Process
•Four Components
•Four-Step Process
•Incremental Compositional QC
Carbon-Isotopic Composition at Four Steps
of a Blending Process
-20
-22
︶
B
D -24
P
V
s
v
o -26
o
/
o
︵
C
-28

3
1
-30
-32
1
2
3
4
Product Step (n)
Step
1
2
3
4
Table 4. d13C of a Series of Products along a Production Pathway
mA
dA
mB
dB
mC
dC
1.00
-30.00
2.50
-20.00
0.50
-12.00
mD
dD
1.50
-27.00
dT
-30.00
-22.86
-21.50
-23.00