Presented at INTERPHEX on March 21-23, 2017. This presentation is an overview of various techniques for performing extractable studies, including protocols of several organizations. It goes into detail in comparing USP requirements to BPOG requirements, showing that in the end, patient safety is demonstrated with limited model solvents and time points.

1. So Many Solvents, So Little Time
Paul F Killian, PhD
MilliporeSigma

2. Agenda
• Introduction
• Overview of Extractables Studies
• Protocols of Various Associations
• Comparison of USP vs BPOG
• Conclusion

3. 1. Introduction
What are Extractables and Leachables?
Oligomers
PVDF, PP, PE
Additives and Degradants
Antioxidants, Slip agents
Residual Solvents
Quantity
and Type
Material of Construction
Contact Fluid Process Conditions

4. Regulatory Requirement
“Equipment shall be constructed so that the surfaces that
contact component, in-process materials, or drug products shall
not be reactive, additive, or absorptive so as to alter the safety,
identity, strength, or purity of the drug product beyond the
official or other established requirements.”

5. Production Issues

6. 2. Overview of Extractable Studies
Extractables: All compounds that could potentially leach from a
material
•Usually obtained by analytical analyses under exaggerated extraction
conditions (a.k.a. “worse-case” normal use conditions)
•From insider knowledge (manufacturer disclosure of ingredients).
Extractables
Leachables
Leachables: What actually leaches from a material into the drug product under a particular
use condition
•Generally a subset of the extractables, at a fraction of the total amount available
•Could be “daughter” extractables; i.e. derived from extractables

7. Reflux Extractions
•Aggressive Solvents
•Components often cut up
•Final Fill Components
•Extraction time <24 hrs
Extractable Study Considerations
Model Solvent Extractions
•Processing Solvents
•Components tested “as is”
•Processing Equipment
•Long Extraction times

8. Static
Extractable Study Considerations
Agitated Recirculated

9. Pre-Flush
Extractable Study Considerations
Sterilization
•Autoclave temperature and cycles
•Gamma vs non-Gamma
0
5
10
15
0 2 4 6 8 10 12 14 16 18 20
Time (minutes)
UVAbsorbanceat214nm
PureFlex (non-Gamma)
PureFlex (Gamma)
RP-HPLC
Non-gamma
Gamma

10. Time points
Extractable Study Considerations
Temperature
45o
C
Room Temp

11. Extractable Study Considerations
Model Solvents
Water for Injection (WFI)
High pH High Salt
Low pH
Water-Alcohol
Surfactant

12. Low
< 1:1
Extractable Study Considerations
High
6:1
Surface Area to Volume Ratio
• More extract for analyses
• Multiple time points from same sample
• Lower detection limits
• Better chance of detecting extractable
compounds

13. Extractable Study Considerations
Assays (Specific)
•Gas Chromatography-Mass Spec (GCMS). Measures the volatile organic compounds
(VOCs) and semivolatile organic compounds (SVOCs) present in the extract.
•Liquid Chromatography with UV and MS detectors (LCUV-MS). Measures the
semivolatile organic compounds (SVOCs) and non-volatile compounds present in the
extract.
•Inductively Coupled Plasma (ICP) Measures the concentration of metals present in the
extract.
•Ion Chromatography (IC). Measures concentrations of anions or cations present in the
extract. (Common extractables include small organic acids and salts).

14. Extractable Study Considerations
Assays (Non-specific)
•Total Organic Carbon (TOC). Measures the total amount of organic material
present. Gives general cleanliness of component.
•Non-Volatile Residue (NVR). Measures the mass of extractables that can not be
evaporated off. Gives general cleanliness of component.
•pH Gives an indication if the extractables will shift the pH of the drug product or
processing solution.
•Conductivity. Gives an indication if the extractables will shift the ionic balance of the
drug product or processing solution.

15. 3. Protocols of Various Organizations

16. ASME-Bioprocesss Equipment (BPE)
Solvents SA to Vol Time Points Temperature Other
WFI
0.1M NaOH
0.1M H3PO4
20% Ethanol
5M NaCl
3:1 30 days 40o
C Un-agitated

17. ASTM WK43975 (Nov 2015 Draft)
Solvents SA to Vol Time Points Temperature Other
WFI (optional)
0.5N NaOH
0.1M H3PO4
50% Ethanol
5M NaCl (optional)
1% PS80 (optional)
6:1 films and
components
1:1 filters
30 min, 1 day,
21 day, 70 day
30 min, 1 day
7 day
40o
C Orbital
Shaker
(50 rpm)

18. Bioprocess Systems Alliance (BPSA)
Solvents SA to Vol Time Points Temperature Other
WFI
High pH (optional)
Low pH (optional)
Ethanol
Surfactant (optional)
> 0.5:1 Bags 30 days
Filter, tubing
1 day
Slightly over
Operating
Temperature
Bags – static or
shaken
Filter -
recirculation

19. USP <661.3> Draft
Solvents SA to Vol Time Points Temperature Other
WFI
0.1N HCl
Acid/Salt, pH 3
Phosphate, pH 10
50% Ethanol
6:1 4 days
1 day, 7 day,
Or 21 day
55o
C
40o
C

20. Biophorum Operations Group (BPOG)
Solvents SA to Vol Time Points Temperature Other
WFI
0.5N NaOH
0.1M H3PO4
50% Ethanol
5M NaCl
1% PS80
6:1 bags,
components
1:1 filters
30 min, 1 day,
21 day, 70 day
30 min, 1 day,
7 days
40o
C Orbital
Shaker
(50 rpm)

21. 4. Comparison USP vs BPOG
Extractables Study for a 10” Filter Capsule
Major Differences:
•Number and type of Solvent
•Number of time points
•Total number of assays

22. Assay WFI 0.1N HCl pH 3 pH 10 50% Ethanol
Samples
per solvent
Time
points
Assays
Subset
GCMS-HS √ √ √ 4 1 12
GCMS-DI √ √ √ 4 1 12
LCMS √ √ √ 4 1 12
Metals √ 4 1 4
TOC √ 4 1 4
Absorbance √ 4 1 4
Acidity /
Alkalinity
√ 4 1 4
USP <661.3> requirements for extractables testing of a filter
Total Assays: 52

23. Assay WFI
0.5N
NaOH
0.1M
H3PO4
50%
Ethanol 5M NaCl 1% PS80
Samples
per solvent
Time
points
Assays
Subset
GCMS-HS √ √ √ √ √ √ 4 3 72
GCMS-DI √ √ √ √ √ √ 4 3 72
LCMS √ √ √ √ √ √ 4 3 72
Metals √ √ 4 3 24
TOC √ √ √ √ 4 3 48
pH √ √ √ √ 4 3 48
Conductivity √ √ √ √ 4 3 48
NVR √ √ 4 3 24
FTIR √ √ 4 3 24
IC √ 4 3 12
BPOG requirements for extractables testing of a filter
Total Assays: 444

24. Solvents
5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0
2 0 0 0 0 0 0
4 0 0 0 0 0 0
6 0 0 0 0 0 0
8 0 0 0 0 0 0
1 e + 0 7
1 . 2 e + 0 7
1 . 4 e + 0 7
1 . 6 e + 0 7
1 . 8 e + 0 7
2 e + 0 7
2 . 2 e + 0 7
2 . 4 e + 0 7
2 . 6 e + 0 7
2 . 8 e + 0 7
3 e + 0 7
3 . 2 e + 0 7
3 . 4 e + 0 7
3 . 6 e + 0 7
3 . 8 e + 0 7
4 e + 0 7
4 . 2 e + 0 7
4 . 4 e + 0 7
4 . 6 e + 0 7
4 . 8 e + 0 7
T im e - - >
A b u n d a n c e
T I C : 0 1 2 8 1 6 0 3 7 . D d a t a . m s
5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0
2 0 0 0 0 0 0
4 0 0 0 0 0 0
6 0 0 0 0 0 0
8 0 0 0 0 0 0
1 e + 0 7
1 . 2 e + 0 7
1 . 4 e + 0 7
1 . 6 e + 0 7
1 . 8 e + 0 7
2 e + 0 7
2 . 2 e + 0 7
2 . 4 e + 0 7
2 . 6 e + 0 7
2 . 8 e + 0 7
3 e + 0 7
3 . 2 e + 0 7
3 . 4 e + 0 7
3 . 6 e + 0 7
3 . 8 e + 0 7
4 e + 0 7
4 . 2 e + 0 7
4 . 4 e + 0 7
4 . 6 e + 0 7
4 . 8 e + 0 7
T im e - - >
A b u n d a n c e
T I C : 0 1 2 8 1 6 0 6 5 . D d a t a . m s
Water
Day 1 Extract
5M NaCl
Day 1 Extract
IS
IS
IS
IS
Hexylene
Glycol
NMP
Hexylene
Glycol
NMP

25. 5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
4 5 0 0 0 0 0
T im e - - >
A b u n d a n c e
T I C : 0 2 1 5 1 6 0 3 2 . D d a t a . m s
5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0
2 0 0 0 0 0 0
4 0 0 0 0 0 0
6 0 0 0 0 0 0
8 0 0 0 0 0 0
1 e + 0 7
1 . 2 e + 0 7
1 . 4 e + 0 7
1 . 6 e + 0 7
1 . 8 e + 0 7
2 e + 0 7
2 . 2 e + 0 7
2 . 4 e + 0 7
2 . 6 e + 0 7
2 . 8 e + 0 7
3 e + 0 7
3 . 2 e + 0 7
3 . 4 e + 0 7
3 . 6 e + 0 7
3 . 8 e + 0 7
4 e + 0 7
4 . 2 e + 0 7
4 . 4 e + 0 7
4 . 6 e + 0 7
4 . 8 e + 0 7
T im e - - >
A b u n d a n c e
T I C : 0 1 2 2 1 6 0 2 7 . D d a t a . m s
Solvents
IS
IS
IS IS50% Ethanol
Day 1 Extract
1% PS80
Day 1 Extract
PS80
peak
PS80
peak
Hexylene
Glycol
Hexylene
Glycol
NMP
NMP

26. Major Extractable Compounds

27. Time Points

28. Time Points

29. Toxicological Profile
N-Methyl Pyrrolidone (NMP)
• Irritant of skin and mucous membranes
• Not Genotoxic
• No skin sensitization potential was found in the guinea pig
• The lowest reported NOAEC was 100 mg/m3
in a 28-day study in rats, after oral
administration
• The lowest reported NOAEL in a 90-day feeding study was 169 or 217 mg/kg/day in male or
female rats, respectively
ICH has established a PDE for NMP of 5.3 mg/day

30. Toxicological Profile
Hexylene Glycol
• Low acute toxicity, regardless of route of exposure:
 LD50, rat, oral > 2,000 mg/kg bw
 Acute intraperitoneal LD50 for the rat and mouse is given as 1,300–1,500 mg/kg
• Low potential to irritate the skin
• Not Genotoxic
• Slightly irritating to the eyes
• After repeat oral exposure for 90 days, a systemic NOAEL of 450 mg/kg/day was found.
• NOAEC after inhalation exposure was 700 mg/m3
in rats
 (7 hours/day, 5 days/week for 11 days)

31. Toxicological Profile
Hexylene Glycol
NOAEC = 700 mg/m3
results in 99.5 mg/kg bw/day
Safety Factors
1. F1 = 5 for rat to human
2. F2 = 10 for intraspecies variability
3. F3 = 10 for short study duration
4. F4 = 1 for no severe toxicity encountered
5. F5 = 1 for NOEL value used
6. F6 = 1 for bioavailability 100%
PDE = 10 mg/day

32. Final Fill Assembly – Sterile Filter

33. Exposure Scenario
Process Parameter Volume
Post Sterilization Flush 2 L
Vials Discarded (10 mL / vial) 10 vials (0.1 L)
Minimum Volume (header bag) 1 L
Total Volume 3.1 L

34. Exposure Scenario
Compound
Maximum
Extractable
(mg/filter)
Minimum
Process Volume
(L)
Final
Concentration
(mg/L)
NMP 5.9 3.1 1.9
Hexylene Glycol 14.8 3.1 4.8

35. Patient Safety Evaluation
Compound
Final
Concentration
(mg/L)
Dosage
(L)
Patient
Exposure
(mg)
PDE
(mg/Day)
NMP 1.9 0.01 0.019 5.3
Hexylene Glycol 4.8 0.01 0.048 10.0

36. 5. Conclusion
• Extractable studies performed for Patient Safety and Production concerns
• There are many different protocols for Extraction Studies
• Multiple solvents with multiple time points results in a lot of data
• Patient Safety is demonstrated using only limited time points / solvents

37. Acknowledgments
Special Thanks to:
Jessica Shea
Dr. Thomas Broschard
George Adams
Thank You