E&L 2017-Final
Continuous Flow Extraction and Analysis of a Single Use Bioprocess System Kevin Rowland, M.S. and Mark Jordi, Ph.D. Analytical Services Division; 200 Gilbert St. Mansfield, MA 02048
Objective
Model Bioprocess System
Single use Bio-processing systems are attractive due to their efficiency and the potential to eliminate complex cleaning procedures and validations. This study was designed to demonstrate an analytical methodology tailored for effective determination of the extractable compounds from a model bioprocess system. A continuous flow extraction was performed in order to investigate the compounds extracted under the conditions most closely representing a use condition. Once compounds had been found and filtered, the effect of standard selection on the number of compounds falling above the analytical evaluation threshold (AET) was investigated.
Bag
Phamaceutical grade
Multilayer Film LDPE Liquid Contact EVA Air Barrier Film
Thermoplastic Elastomer Ultra-pure fluid Processing applications
Extraction Vehicle:
Nylon Filter Polypropylene Housing Ethylene propylene O-ring
Filter
Analytical Methodology
Saline- 137mM NaCl, 2.7mM KCl 10 mM Phosphate Buffer (pH 7.4), 300 mL
Hydrophilic disk and holder
Scouting Volatiles System and Component Extracts
Tubing
Bioprocess
FTIR UV-VIS Grav. TOC Dynamic
Headspace GCMS
Data Analysis and Identification
GC/Q-TOF
Semi-Volatiles
Extraction Conditions: Statistical Analysis
LC/Q-TOF
Non-Volatiles
•Recursive Feature Finding •Fold Change and Abundance Filtering
ICP-MS
Elemental
Library Searches •Proprietary Databases and Libraries •NIST, Wiley
Continuous recirculation with a perastaltic pump; 72 hours; 37°C
Unknown ID •Formula Generation •MS/MS Analysis
Scouting Analyses
Elemental Analysis 3.0
Method Blank Extract
ng/Device µg/Day
Spectroscopy
2.078mg
ICP-MS
2.607mg
2.0
UV absorption (mAU)
Tubing Bag Filter Continuous Flow
10.57mg
Values are per device
Total Organic Carbon
2.5
1.5
1.0
0.5
0.0
-0.5
0.244mg
200
400
600 Wavelength (nm)
800
As
4.5 Co 0.018
Cd
0.6 Ni 0.002
< IDL
Sb
77 0.31
Hg
5.7 Cr 0.023
12 Ba 0.047
< IDL
Pb
21 Na 0.084
Matrix 350,000 ng/mL
25 Mo 0.098
7.8 0.031
Values Based on 500 doses per device, 2 doses per day < IDL - Less than the instrumental detection limit
1000
Organic Identification Method Blank Extract
1.25
2
0.75
0.5
1
1
2
4
3
5
0.25
0
x107 Method Blank Extract
Method Blank Extract
1
Agilent 6545 LC/Q-TOF
LC/Q-TOF
3
ESI-
0
1
2
3
4
5
6
7
8
9
1
2
Counts vs. Acquisition Time (min)
3
4
5
6
7
8
9
Counts vs. Acquisition Time (min)
2
1
Agilent 7200 GC/Q-TOF
ESI+
x10 5
GC/Q-TOF
x10
6
0
5
10
15
20
25
30
Counts vs. Acquisition Time (min)
Analytical Evaluation Threshold AET=
=
Accounting for the extraction performed, the intstrumental limit is:
=37.5 µg/Device
Erucylamide
Analytical
37.5 µg/Device ÷ 300 mL = 0.125 µg/mL
Norwood D. The Analytical Evaluation Threshold (AET) and Its Relationship to Safety Thresholds, Leachables and Extractables Handbook (2012): 59-78. PQRI.org.
Response factor Database
Standard
Number of compounds observed Above AET depends on Standard Selected Total Extract Unique Compounds Detected Filtered Using Erucylamide Standard
60
Filtered Using Response Factor Database %RSD = 76%
59 1
Irganox 1141 1
2
3
4
5
Conclusions
1
Filtered Using Irganox 1141 Standard
Approx. AET Response Cut-off
A wide array of analytical techniques are required for thorough identification of system extractables. Extraction methodology effects the quantity of extractables observed. The analytical standard used to relate AET concentration to instrument response has a strong effect on the response cut-off. PQRI recommendation of adjusting the AET by the greater of 50% or the %RSD of the relative response factor database represents good compromise.
www.jordilabs.com 508-966-1301
Objective
Model Bioprocess System
Single use Bio-processing systems are attractive due to their efficiency and the potential to eliminate complex cleaning procedures and validations. This study was designed to demonstrate an analytical methodology tailored for effective determination of the extractable compounds from a model bioprocess system. A continuous flow extraction was performed in order to investigate the compounds extracted under the conditions most closely representing a use condition. Once compounds had been found and filtered, the effect of standard selection on the number of compounds falling above the analytical evaluation threshold (AET) was investigated.
Bag
Phamaceutical grade
Multilayer Film LDPE Liquid Contact EVA Air Barrier Film
Thermoplastic Elastomer Ultra-pure fluid Processing applications
Extraction Vehicle:
Nylon Filter Polypropylene Housing Ethylene propylene O-ring
Filter
Analytical Methodology
Saline- 137mM NaCl, 2.7mM KCl 10 mM Phosphate Buffer (pH 7.4), 300 mL
Hydrophilic disk and holder
Scouting Volatiles System and Component Extracts
Tubing
Bioprocess
FTIR UV-VIS Grav. TOC Dynamic
Headspace GCMS
Data Analysis and Identification
GC/Q-TOF
Semi-Volatiles
Extraction Conditions: Statistical Analysis
LC/Q-TOF
Non-Volatiles
•Recursive Feature Finding •Fold Change and Abundance Filtering
ICP-MS
Elemental
Library Searches •Proprietary Databases and Libraries •NIST, Wiley
Continuous recirculation with a perastaltic pump; 72 hours; 37°C
Unknown ID •Formula Generation •MS/MS Analysis
Scouting Analyses
Elemental Analysis 3.0
Method Blank Extract
ng/Device µg/Day
Spectroscopy
2.078mg
ICP-MS
2.607mg
2.0
UV absorption (mAU)
Tubing Bag Filter Continuous Flow
10.57mg
Values are per device
Total Organic Carbon
2.5
1.5
1.0
0.5
0.0
-0.5
0.244mg
200
400
600 Wavelength (nm)
800
As
4.5 Co 0.018
Cd
0.6 Ni 0.002
< IDL
Sb
77 0.31
Hg
5.7 Cr 0.023
12 Ba 0.047
< IDL
Pb
21 Na 0.084
Matrix 350,000 ng/mL
25 Mo 0.098
7.8 0.031
Values Based on 500 doses per device, 2 doses per day < IDL - Less than the instrumental detection limit
1000
Organic Identification Method Blank Extract
1.25
2
0.75
0.5
1
1
2
4
3
5
0.25
0
x107 Method Blank Extract
Method Blank Extract
1
Agilent 6545 LC/Q-TOF
LC/Q-TOF
3
ESI-
0
1
2
3
4
5
6
7
8
9
1
2
Counts vs. Acquisition Time (min)
3
4
5
6
7
8
9
Counts vs. Acquisition Time (min)
2
1
Agilent 7200 GC/Q-TOF
ESI+
x10 5
GC/Q-TOF
x10
6
0
5
10
15
20
25
30
Counts vs. Acquisition Time (min)
Analytical Evaluation Threshold AET=
=
Accounting for the extraction performed, the intstrumental limit is:
=37.5 µg/Device
Erucylamide
Analytical
37.5 µg/Device ÷ 300 mL = 0.125 µg/mL
Norwood D. The Analytical Evaluation Threshold (AET) and Its Relationship to Safety Thresholds, Leachables and Extractables Handbook (2012): 59-78. PQRI.org.
Response factor Database
Standard
Number of compounds observed Above AET depends on Standard Selected Total Extract Unique Compounds Detected Filtered Using Erucylamide Standard
60
Filtered Using Response Factor Database %RSD = 76%
59 1
Irganox 1141 1
2
3
4
5
Conclusions
1
Filtered Using Irganox 1141 Standard
Approx. AET Response Cut-off
A wide array of analytical techniques are required for thorough identification of system extractables. Extraction methodology effects the quantity of extractables observed. The analytical standard used to relate AET concentration to instrument response has a strong effect on the response cut-off. PQRI recommendation of adjusting the AET by the greater of 50% or the %RSD of the relative response factor database represents good compromise.
www.jordilabs.com 508-966-1301
