Case Study of an Unlikely Leachable in a High Potency ...
Case Study of an Unlikely Leachable in a High Potency Pharmaceutical Product By Jaime Marach, Ph.D. Senior Scientist, R&D AAPS 2014 Annual Meeting November 6, 2014 1
Outline • Dr. Dennis Jenke: overview of what an extractable or leachable is, chemical and safety assessment, case studies… • The Case Study of an OOS impurity in a drug product on stability • OOS Characterization of leachable • Determination of leachable source • Environmental presence and toxicity of leachable 2
The Case of the Out of Specification (OOS) Impurity in Drug Product Stability Testing • A high potency drug product was manufactured in vials and placed on stability • 1 of 3 lots manufactured had an Out of Specification (OOS) known impurity result at 12 months • Typical Limit of Quantitation (LOQ) is 0.05% - 0.10% for a high potency product (e.g., ng or μg level), can reach ppb level of impurities will detect impurities, contaminants, etc. that won’t usually see on stability, when compared with less potent products/less sensitive methods
3
12 Month Stability Data
0.006 0.004 0.002
Unk @ RRT 1.15 - 18.428
0.008
Unk @ RRT 1.10 - 17.122
Rel Compd B - 12.070
AU
0.010
16.839
0.012
16.176
0.014
14.969
0.016
Unk @ RRT 0.90 - 14.331
0.018
Rel Compd C - 13.600
Known impurity at RT 12.07 min
0.020
Unk @ RRT 1.04 - 16.490
0.022
Paricalcitol - 15.642
Rel Compd D - 15.244
Identification Solution
0.000 -0.002 -0.004 -0.006 9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50
13.00
13.50
14.00 Minutes
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
4
12 Month Stability Data Placebo Control 0.016
0.010
Unk @ RRT 1.44 - 22.746 Unk @ RRT 1.46 - 23.120
Rel Compd C - 13.375
0.002
Rel Compd B - 12.134
0.004
Placebo peak 2 - 7.497
0.006
Placebo peak 3 - 8.096
Placebo peak 1 - 6.533
AU
0.008
system peak - 28.005
0.012
Placebo peak 4 - 31.937
0.000001% At 12.13 min
0.014
0.000
-0.002 2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
Minutes
• RP-HPLC impurity method • Region of interest: ~12 minutes. • “No” peak in placebo 5
12 Month Stability Data Paricalcitol - 15.743
Inverted Stability Sample 0.032
Impurity at 12.21 min, 0.1% (ID solution was 12.07 min)
0.030 0.028 0.026 0.024 0.022
0.018
0.002 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00 18.00 Minutes
19.00
20.00
21.00
22.00
23.00
system peak - 28.089
Unk @ RRT 1.46 - 23.206
0.004
Rel Compd E - 21.304
0.006
Unk @ RRT 1.04 - 16.530
0.008
Unk @ RRT 0.90 - 14.197
0.010
Placebo peak 3 - 8.231
0.012
Rel Compd B - 12.211
Unk @ RRT 0.65 - 10.384
0.014
Unk @ RRT 1.13 - 17.793 Unk @ RRT 1.15 - 18.123
0.016
Placebo peak 1 - 6.787
AU
0.020
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
6
12 Month Stability Data
0.022 0.020
0.016
Placebo peak 2 - 7.558
0.012 0.010 0.008 0.006 0.004 0.002
Unk @ RRT 0.65 - 10.375
0.014
Placebo peak 3 - 8.195
AU
0.018
Placebo peak 4 - 32.071
0.024
Unk @ RRT 1.42 - 22.458 Unk @ RRT 1.44 - 22.757 Unk @ RRT 1.46 - 23.194
0.026
Rel Compd E - 21.296
0.028
Unk @ RRT 1.04 - 16.519
Impurity at 12.19 min, 2.6% (ID solution was 12.07 min)
0.030
Rel Compd B - 12.191
0.032
Unk @ RRT 1.10 - 17.319 Unk @ RRT 1.13 - 17.777 Unk @ RRT 1.15 - 18.056
Paricalcitol - 15.732
Upright Stability Sample
0.000 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00 18.00 Minutes
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
7
The Case of the Out of Specification (OOS) Impurity in Drug Product Stability Testing • The known impurity was not expected to be a major degradation product • No apparent trend at 12M shelf-life for upright vs. inverted vials • Hypotheses: – Lab error, contamination, instrument error – Rouging: water used in manufacturing, iron oxide source, catalysis – Glass vials: leachables, catalysis with glass surfaces • No plastic vial and no concern of inks on labels traveling through plastic
– Stoppers: leachables – Suspicion of co-elution of another impurity with the known impurity
8
LC-MS Investigation • Further aging of stability samples begins to show higher levels of the unknown and higher preponderance of vials with the unknown in the Inverted orientation. • Stopper is suspected. • Stopper is 13 mm, coated (fluorotec). • Examine UV spectra and contact LC-MS contractor for ID work with hypothesis…
9
LC-MS Investigation Stability Sample
Active
10
LC-MS Investigation Benzophenone Standard LC-MS Match with Benzo standard
11
Demonstration of Source of Benzophenone • Due to larger amount of benzophenone in inverted vials, stoppers are suspected • Stoppers are coated • Contact stopper manufacturer regarding stopper formulation • Stopper manufacturer states that benzophenone is not used 12
Demonstration of Source of Benzophenone • Glass vials are not possible to be a source • Excipient source? • Perform extraction studies on stoppers by refluxing in placebo • Typical reflux would also include organic solvent • Benzophenone is known to be soluble in hexane 13
Reflux Experiment
14
Reflux Experiment Stoppers were removed from actual stability vials, rinsed, dried and cut into 1/4ths and placed into a round bottom with placebo
Placebo 15
Reflux Experiment The cut stoppers in the round bottom flask are refluxed with placebo for 6 hours, analyze
16
Reflux Experiment Diluent Blank 0.0024
0.0022
0.0020
0.0018
0.0016
No benzophenone peak
AU
0.0014
18.174
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
17
0.032 0.030
3.517
Reflux Experiment Refluxed Placebo, No Stoppers 0.028
No benzophenone peak
0.026 0.024 0.022 0.020 0.018
0.014
4.021
0.016
0.012
0.002 0.000
18.209 19.239
0.004
13.653
0.006
3.775 3.923 4.147 4.340 4.256 4.483 5.054 5.175 5.299 5.403 6.606 7.649 7.924
0.008
-0.002 -0.004 -0.006
3.350
AU
0.010
-0.008 -0.010 -0.012 -0.014 -0.016 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
18
Reflux Experiment Placebo – No Reflux 0.034
0.030
No benzophenone peak
3.507
0.032
0.028 0.026 0.024 0.022 0.020 0.018 0.016 0.014
0.004 0.002 0.000
18.202 19.234
0.006
8.037
0.008
3.674 3.854 3.937 4.0794.209 4.516 4.401 4.649 4.811 4.986 5.068 5.165 5.424
0.010
-0.002 -0.004 -0.006
3.350
AU
0.012
-0.008 -0.010 -0.012 -0.014 -0.016 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
19
Reflux Experiment Benzophenone in Diluent Std 0.034
12.081
0.032 0.030 0.028 0.026 0.024 0.022 0.020
0.016 0.014 0.012 0.010 0.008 0.006
0.002
18.155
0.004
3.466
AU
0.018
0.000
0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
20
-0.004
-0.006
3.357
0.010
0.008
0.006
0.004
0.002
0.000
2.00 4.00 6.00 8.00 10.00
0.018
0.016
21.147
0.030
12.00 Minutes 14.00 16.00 18.00 20.00
21.821
0.032
16.437
0.034
17.195 17.623 17.995 18.209 18.714 18.971 19.241 19.512 20.006
12.179
10.369
0.012
12.552 12.753 13.174 13.637 14.126 14.693 14.886 15.211 15.561 15.946
11.627
10.654
8.829 9.364 9.719
0.036
3.532
0.038
7.015 7.319 7.508 8.069 8.310
3.625 3.732 3.851 4.1723.984 4.4134.309 4.500 4.566 4.916 5.026 5.146 5.408 5.540 5.806 6.100 6.249
AU
Reflux Experiment Refluxed Stoppers from Stability Sample Vials in Placebo 0.040
0.028
0.026
0.024
0.022
0.020
0.014
-0.002
-0.008
-0.010
-0.012
-0.014
B.Roth
22.00
21
Reflux Experiment Refluxed Stoppers from Stability Sample Vials in Placebo, Zoom 0.0125 0.0120 0.0115 0.0110 0.0105 0.0100 0.0095
12.179
0.0090 0.0085 0.0080
10.369
0.0075 0.0070
0.0060 0.0055 0.0050 0.0045
0.0010
14.886 14.693
0.0015
11.627
0.0020
14.126
0.0025
12.753
9.719
10.654
0.0030
13.637
12.552
0.0035
13.174
0.0040
9.364
RT match with benzo standard
AU
0.0065
0.0005 0.0000
B.Roth
-0.0005 9.50
10.00
10.50
11.00
11.50
12.00 Minutes
12.50
13.00
13.50
14.00
14.50
22
UV Spectrum Benzophenone 0.2 g/mL 12.087 Extracted 0.180 0.170 0.160 0.150 0.140 0.130 0.120 0.110
AU
0.100 0.090 0.080 0.070 0.060 0.050 0.040
253.0 0.030 0.020 0.010
335.0 0.000 190.00
200.00
210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00 nm
310.00
320.00
330.00
340.00
350.00
360.00
B.Roth
370.00
380.00
390.00
400.00
23
UV Spectrum Refluxed stoppers in placebo (stoppers obtained from product vials) 12.174 Peak 1 0.017
191.0
0.016 0.015
UV spectrum match with benzo standard
0.014 0.013 0.012 0.011 0.010
AU
0.009 0.008 0.007
253.0
0.006 0.005 0.004 0.003 0.002 0.001
335.0
370.0
383.0
0.000
190.00
200.00
210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00 nm
310.00
320.00
330.00
340.00
350.00
360.00
370.00
B.Roth
380.00
390.00
400.00
24
OOS is Benzophenone • UV spectrum, Retention Time, and LC-MS match between OOS peak and benzophenone • Reflux experiment traces it to stoppers (even though stopper manufacturer says it’s not possible)
25
What is Benzophenone? • MW 182.22 C13H10O • Used as a photoinitiator, a fragrance enhancer, an ultraviolet curing agent, and, occasionally, as a flavor ingredient; also manufacture of insecticides, agricultural chemicals, and pharmaceuticals and is an additive for plastics, coatings, and adhesives. • Pharma: common leachable in UV-based inks, labels, and coatings • Strong chromophore 26
What is Benzophenone? • Benzophenone can become a leachable because it is not entirely consumed during the UV curing process for coatings (e.g., stopper coating) • Thus, free benzophenone may be leachable upon contact with various liquids over time • May migrate to the coating surface over time
Fan et al., US Patent 5,900,472
27
What is the Metabolism of Benzophenone?
• Target organ for toxicity in rodents: liver or kidney • Non-genotoxic IARC Monograph: Benzophenone
28
There’s Benzophenone in my WHAT? • Benzophenone is used as an ultraviolet (UV)curing agent in sunglasses • Prevent UV light from damaging scents and colors in products such as perfumes and soaps. • UV blocker added to plastic packaging, which allows manufacturers to package their products in clear glass or plastic rather than opaque or dark packaging. • Laundry and household cleaning products
IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
29
There’s Benzophenone in my WHAT? • Benzophenone was reported to occur naturally in wine grapes at concentrations of 0.08–0.13 ppm [mg/kg], mainly in muscat grapes • Food products range from 0.57 ppm [mg/kg] benzophenone in nonalcoholic beverages to 3.27 ppm [mg/kg] in frozen dairy products • May also be an ingredient in baked goods, soft candy, gelatins, and puddings IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
30
Benzophenone in Cereal
31
Benzophenone in Cereal • Photoinitator for Inks: 4-Methylbenzophenone, in combination with, or as a replacement to benzophenone, is used for UV-cured printing inks and lacquers applied on package surfaces, mainly cardboard boxes. • Migration: Given their volatility, both substances may migrate into the package and contaminate even solid foodstuffs. – Easily migrates through polypropylene film; aluminium and multilayer materials inhibit migration efficiently. • Internal plastic bags: used as a barrier against moisture are not always effective • Recycling: Most commonly used raw material for paperboard is recycled, which thereafter may contains photo-initiators, including benzophenone. – Direct contact with dry foodstuffs, such as flour and pasta, but also with fast-food items, i.e. foodstuffs with a short duration of contact, such as pizzas. Normally, a functional barrier, e.g. plastic or aluminium foil, is used between fatty or aqueous foodstuffs and the recycled material to avoid direct contact. EFSA statement on the presence of 4-methylbenzophenone found in breakfast cereals, 2009 IARC Monograph: Benzophenone
32
What is the Toxicity of Benzophenone? • Based on the use of benzophenone as an additive in fragrances, cosmetics, toiletries, pharmaceuticals, insecticides, and flavor ingredients, consumer exposure may be significant • Listed in the EU register of chemically defined flavorings • European Food Safety Authority (EFSA) estimated dietary exposure in the USA to be 11 μg per capita per day. • Tolerable Daily Intake: Scientific Committee on Food had established for benzophenone and 4hydroxybenzophenone a group TDI of 0.01 mg/kg body weight Example: 0.01 mg/kg * 70 kg = 0.7 mg intake IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
33
What is the Toxicity of Benzophenone? Tolerable Daily Intake Example: 0.01 mg/kg * 70 kg = 0.7 mg intake In high potency drug product, concentration is approximately 0.001 g/mL (small)
IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
34
Conclusions • Benzophenone, a common curing agent, was found to be coeluting with a known impurity in a stability sample • Benzophenone was extracted from the product stoppers • A review of literature was performed to understand the everyday exposure and toxicity • A control strategy for the drug product must be put in place
35
Questions?
36
37
Certain Stored Placebos Contain Contaminant of Benzophenone Placebo Blank 0.0100 0.0095 0.0090 0.0085 0.0080 0.0075 0.0070 0.0065 0.0060
Benzophenone - 12.197
AU
0.0055 0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00 10.00 Minutes
11.00
12.00
13.00
14.00
15.00
16.00
38
Identification by RT Placebo Blank 0.0105 0.0100
Benzophenone peak in stored excipient component
0.0095 0.0090 0.0085 0.0080 0.0075 0.0070 0.0065
Benzophenone - 12.197
0.0060
AU
0.0055 0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000
2.00
4.00
6.00
8.00
10.00
12.00
14.00 Minutes
16.00
18.00
20.00
22.00
24.00
26.00
39
Identification by RT Placebo – Fresh, Different Manufacturer 0.0100 0.0095 0.0090 0.0085
No benzophenone peak
0.0080 0.0075 0.0070 0.0065 0.0060 0.0055
AU
0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 -0.0005 2.00
4.00
6.00
8.00
10.00
12.00
14.00 Minutes
16.00
18.00
20.00
22.00
24.00
26.00
40
Outline • Dr. Dennis Jenke: overview of what an extractable or leachable is, chemical and safety assessment, case studies… • The Case Study of an OOS impurity in a drug product on stability • OOS Characterization of leachable • Determination of leachable source • Environmental presence and toxicity of leachable 2
The Case of the Out of Specification (OOS) Impurity in Drug Product Stability Testing • A high potency drug product was manufactured in vials and placed on stability • 1 of 3 lots manufactured had an Out of Specification (OOS) known impurity result at 12 months • Typical Limit of Quantitation (LOQ) is 0.05% - 0.10% for a high potency product (e.g., ng or μg level), can reach ppb level of impurities will detect impurities, contaminants, etc. that won’t usually see on stability, when compared with less potent products/less sensitive methods
3
12 Month Stability Data
0.006 0.004 0.002
Unk @ RRT 1.15 - 18.428
0.008
Unk @ RRT 1.10 - 17.122
Rel Compd B - 12.070
AU
0.010
16.839
0.012
16.176
0.014
14.969
0.016
Unk @ RRT 0.90 - 14.331
0.018
Rel Compd C - 13.600
Known impurity at RT 12.07 min
0.020
Unk @ RRT 1.04 - 16.490
0.022
Paricalcitol - 15.642
Rel Compd D - 15.244
Identification Solution
0.000 -0.002 -0.004 -0.006 9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50
13.00
13.50
14.00 Minutes
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
4
12 Month Stability Data Placebo Control 0.016
0.010
Unk @ RRT 1.44 - 22.746 Unk @ RRT 1.46 - 23.120
Rel Compd C - 13.375
0.002
Rel Compd B - 12.134
0.004
Placebo peak 2 - 7.497
0.006
Placebo peak 3 - 8.096
Placebo peak 1 - 6.533
AU
0.008
system peak - 28.005
0.012
Placebo peak 4 - 31.937
0.000001% At 12.13 min
0.014
0.000
-0.002 2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
Minutes
• RP-HPLC impurity method • Region of interest: ~12 minutes. • “No” peak in placebo 5
12 Month Stability Data Paricalcitol - 15.743
Inverted Stability Sample 0.032
Impurity at 12.21 min, 0.1% (ID solution was 12.07 min)
0.030 0.028 0.026 0.024 0.022
0.018
0.002 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00 18.00 Minutes
19.00
20.00
21.00
22.00
23.00
system peak - 28.089
Unk @ RRT 1.46 - 23.206
0.004
Rel Compd E - 21.304
0.006
Unk @ RRT 1.04 - 16.530
0.008
Unk @ RRT 0.90 - 14.197
0.010
Placebo peak 3 - 8.231
0.012
Rel Compd B - 12.211
Unk @ RRT 0.65 - 10.384
0.014
Unk @ RRT 1.13 - 17.793 Unk @ RRT 1.15 - 18.123
0.016
Placebo peak 1 - 6.787
AU
0.020
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
6
12 Month Stability Data
0.022 0.020
0.016
Placebo peak 2 - 7.558
0.012 0.010 0.008 0.006 0.004 0.002
Unk @ RRT 0.65 - 10.375
0.014
Placebo peak 3 - 8.195
AU
0.018
Placebo peak 4 - 32.071
0.024
Unk @ RRT 1.42 - 22.458 Unk @ RRT 1.44 - 22.757 Unk @ RRT 1.46 - 23.194
0.026
Rel Compd E - 21.296
0.028
Unk @ RRT 1.04 - 16.519
Impurity at 12.19 min, 2.6% (ID solution was 12.07 min)
0.030
Rel Compd B - 12.191
0.032
Unk @ RRT 1.10 - 17.319 Unk @ RRT 1.13 - 17.777 Unk @ RRT 1.15 - 18.056
Paricalcitol - 15.732
Upright Stability Sample
0.000 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00 18.00 Minutes
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
7
The Case of the Out of Specification (OOS) Impurity in Drug Product Stability Testing • The known impurity was not expected to be a major degradation product • No apparent trend at 12M shelf-life for upright vs. inverted vials • Hypotheses: – Lab error, contamination, instrument error – Rouging: water used in manufacturing, iron oxide source, catalysis – Glass vials: leachables, catalysis with glass surfaces • No plastic vial and no concern of inks on labels traveling through plastic
– Stoppers: leachables – Suspicion of co-elution of another impurity with the known impurity
8
LC-MS Investigation • Further aging of stability samples begins to show higher levels of the unknown and higher preponderance of vials with the unknown in the Inverted orientation. • Stopper is suspected. • Stopper is 13 mm, coated (fluorotec). • Examine UV spectra and contact LC-MS contractor for ID work with hypothesis…
9
LC-MS Investigation Stability Sample
Active
10
LC-MS Investigation Benzophenone Standard LC-MS Match with Benzo standard
11
Demonstration of Source of Benzophenone • Due to larger amount of benzophenone in inverted vials, stoppers are suspected • Stoppers are coated • Contact stopper manufacturer regarding stopper formulation • Stopper manufacturer states that benzophenone is not used 12
Demonstration of Source of Benzophenone • Glass vials are not possible to be a source • Excipient source? • Perform extraction studies on stoppers by refluxing in placebo • Typical reflux would also include organic solvent • Benzophenone is known to be soluble in hexane 13
Reflux Experiment
14
Reflux Experiment Stoppers were removed from actual stability vials, rinsed, dried and cut into 1/4ths and placed into a round bottom with placebo
Placebo 15
Reflux Experiment The cut stoppers in the round bottom flask are refluxed with placebo for 6 hours, analyze
16
Reflux Experiment Diluent Blank 0.0024
0.0022
0.0020
0.0018
0.0016
No benzophenone peak
AU
0.0014
18.174
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
17
0.032 0.030
3.517
Reflux Experiment Refluxed Placebo, No Stoppers 0.028
No benzophenone peak
0.026 0.024 0.022 0.020 0.018
0.014
4.021
0.016
0.012
0.002 0.000
18.209 19.239
0.004
13.653
0.006
3.775 3.923 4.147 4.340 4.256 4.483 5.054 5.175 5.299 5.403 6.606 7.649 7.924
0.008
-0.002 -0.004 -0.006
3.350
AU
0.010
-0.008 -0.010 -0.012 -0.014 -0.016 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
18
Reflux Experiment Placebo – No Reflux 0.034
0.030
No benzophenone peak
3.507
0.032
0.028 0.026 0.024 0.022 0.020 0.018 0.016 0.014
0.004 0.002 0.000
18.202 19.234
0.006
8.037
0.008
3.674 3.854 3.937 4.0794.209 4.516 4.401 4.649 4.811 4.986 5.068 5.165 5.424
0.010
-0.002 -0.004 -0.006
3.350
AU
0.012
-0.008 -0.010 -0.012 -0.014 -0.016 0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
B.Roth
19
Reflux Experiment Benzophenone in Diluent Std 0.034
12.081
0.032 0.030 0.028 0.026 0.024 0.022 0.020
0.016 0.014 0.012 0.010 0.008 0.006
0.002
18.155
0.004
3.466
AU
0.018
0.000
0.00
5.00
10.00
15.00
20.00
25.00 Minutes
30.00
35.00
40.00
45.00
20
-0.004
-0.006
3.357
0.010
0.008
0.006
0.004
0.002
0.000
2.00 4.00 6.00 8.00 10.00
0.018
0.016
21.147
0.030
12.00 Minutes 14.00 16.00 18.00 20.00
21.821
0.032
16.437
0.034
17.195 17.623 17.995 18.209 18.714 18.971 19.241 19.512 20.006
12.179
10.369
0.012
12.552 12.753 13.174 13.637 14.126 14.693 14.886 15.211 15.561 15.946
11.627
10.654
8.829 9.364 9.719
0.036
3.532
0.038
7.015 7.319 7.508 8.069 8.310
3.625 3.732 3.851 4.1723.984 4.4134.309 4.500 4.566 4.916 5.026 5.146 5.408 5.540 5.806 6.100 6.249
AU
Reflux Experiment Refluxed Stoppers from Stability Sample Vials in Placebo 0.040
0.028
0.026
0.024
0.022
0.020
0.014
-0.002
-0.008
-0.010
-0.012
-0.014
B.Roth
22.00
21
Reflux Experiment Refluxed Stoppers from Stability Sample Vials in Placebo, Zoom 0.0125 0.0120 0.0115 0.0110 0.0105 0.0100 0.0095
12.179
0.0090 0.0085 0.0080
10.369
0.0075 0.0070
0.0060 0.0055 0.0050 0.0045
0.0010
14.886 14.693
0.0015
11.627
0.0020
14.126
0.0025
12.753
9.719
10.654
0.0030
13.637
12.552
0.0035
13.174
0.0040
9.364
RT match with benzo standard
AU
0.0065
0.0005 0.0000
B.Roth
-0.0005 9.50
10.00
10.50
11.00
11.50
12.00 Minutes
12.50
13.00
13.50
14.00
14.50
22
UV Spectrum Benzophenone 0.2 g/mL 12.087 Extracted 0.180 0.170 0.160 0.150 0.140 0.130 0.120 0.110
AU
0.100 0.090 0.080 0.070 0.060 0.050 0.040
253.0 0.030 0.020 0.010
335.0 0.000 190.00
200.00
210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00 nm
310.00
320.00
330.00
340.00
350.00
360.00
B.Roth
370.00
380.00
390.00
400.00
23
UV Spectrum Refluxed stoppers in placebo (stoppers obtained from product vials) 12.174 Peak 1 0.017
191.0
0.016 0.015
UV spectrum match with benzo standard
0.014 0.013 0.012 0.011 0.010
AU
0.009 0.008 0.007
253.0
0.006 0.005 0.004 0.003 0.002 0.001
335.0
370.0
383.0
0.000
190.00
200.00
210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00 nm
310.00
320.00
330.00
340.00
350.00
360.00
370.00
B.Roth
380.00
390.00
400.00
24
OOS is Benzophenone • UV spectrum, Retention Time, and LC-MS match between OOS peak and benzophenone • Reflux experiment traces it to stoppers (even though stopper manufacturer says it’s not possible)
25
What is Benzophenone? • MW 182.22 C13H10O • Used as a photoinitiator, a fragrance enhancer, an ultraviolet curing agent, and, occasionally, as a flavor ingredient; also manufacture of insecticides, agricultural chemicals, and pharmaceuticals and is an additive for plastics, coatings, and adhesives. • Pharma: common leachable in UV-based inks, labels, and coatings • Strong chromophore 26
What is Benzophenone? • Benzophenone can become a leachable because it is not entirely consumed during the UV curing process for coatings (e.g., stopper coating) • Thus, free benzophenone may be leachable upon contact with various liquids over time • May migrate to the coating surface over time
Fan et al., US Patent 5,900,472
27
What is the Metabolism of Benzophenone?
• Target organ for toxicity in rodents: liver or kidney • Non-genotoxic IARC Monograph: Benzophenone
28
There’s Benzophenone in my WHAT? • Benzophenone is used as an ultraviolet (UV)curing agent in sunglasses • Prevent UV light from damaging scents and colors in products such as perfumes and soaps. • UV blocker added to plastic packaging, which allows manufacturers to package their products in clear glass or plastic rather than opaque or dark packaging. • Laundry and household cleaning products
IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
29
There’s Benzophenone in my WHAT? • Benzophenone was reported to occur naturally in wine grapes at concentrations of 0.08–0.13 ppm [mg/kg], mainly in muscat grapes • Food products range from 0.57 ppm [mg/kg] benzophenone in nonalcoholic beverages to 3.27 ppm [mg/kg] in frozen dairy products • May also be an ingredient in baked goods, soft candy, gelatins, and puddings IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
30
Benzophenone in Cereal
31
Benzophenone in Cereal • Photoinitator for Inks: 4-Methylbenzophenone, in combination with, or as a replacement to benzophenone, is used for UV-cured printing inks and lacquers applied on package surfaces, mainly cardboard boxes. • Migration: Given their volatility, both substances may migrate into the package and contaminate even solid foodstuffs. – Easily migrates through polypropylene film; aluminium and multilayer materials inhibit migration efficiently. • Internal plastic bags: used as a barrier against moisture are not always effective • Recycling: Most commonly used raw material for paperboard is recycled, which thereafter may contains photo-initiators, including benzophenone. – Direct contact with dry foodstuffs, such as flour and pasta, but also with fast-food items, i.e. foodstuffs with a short duration of contact, such as pizzas. Normally, a functional barrier, e.g. plastic or aluminium foil, is used between fatty or aqueous foodstuffs and the recycled material to avoid direct contact. EFSA statement on the presence of 4-methylbenzophenone found in breakfast cereals, 2009 IARC Monograph: Benzophenone
32
What is the Toxicity of Benzophenone? • Based on the use of benzophenone as an additive in fragrances, cosmetics, toiletries, pharmaceuticals, insecticides, and flavor ingredients, consumer exposure may be significant • Listed in the EU register of chemically defined flavorings • European Food Safety Authority (EFSA) estimated dietary exposure in the USA to be 11 μg per capita per day. • Tolerable Daily Intake: Scientific Committee on Food had established for benzophenone and 4hydroxybenzophenone a group TDI of 0.01 mg/kg body weight Example: 0.01 mg/kg * 70 kg = 0.7 mg intake IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
33
What is the Toxicity of Benzophenone? Tolerable Daily Intake Example: 0.01 mg/kg * 70 kg = 0.7 mg intake In high potency drug product, concentration is approximately 0.001 g/mL (small)
IARC Monograph: Benzophenone National Toxicology Program Toxicity Report Series Number 61 The EFSA Journal (2009) 1104, 1-30 SCIENTIFIC OPINION, Toxicological Evaluation of Benzophenone
34
Conclusions • Benzophenone, a common curing agent, was found to be coeluting with a known impurity in a stability sample • Benzophenone was extracted from the product stoppers • A review of literature was performed to understand the everyday exposure and toxicity • A control strategy for the drug product must be put in place
35
Questions?
36
37
Certain Stored Placebos Contain Contaminant of Benzophenone Placebo Blank 0.0100 0.0095 0.0090 0.0085 0.0080 0.0075 0.0070 0.0065 0.0060
Benzophenone - 12.197
AU
0.0055 0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00 10.00 Minutes
11.00
12.00
13.00
14.00
15.00
16.00
38
Identification by RT Placebo Blank 0.0105 0.0100
Benzophenone peak in stored excipient component
0.0095 0.0090 0.0085 0.0080 0.0075 0.0070 0.0065
Benzophenone - 12.197
0.0060
AU
0.0055 0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000
2.00
4.00
6.00
8.00
10.00
12.00
14.00 Minutes
16.00
18.00
20.00
22.00
24.00
26.00
39
Identification by RT Placebo – Fresh, Different Manufacturer 0.0100 0.0095 0.0090 0.0085
No benzophenone peak
0.0080 0.0075 0.0070 0.0065 0.0060 0.0055
AU
0.0050 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 -0.0005 2.00
4.00
6.00
8.00
10.00
12.00
14.00 Minutes
16.00
18.00
20.00
22.00
24.00
26.00
40
