Extractables Evaluation of a Single-Use Bioprocess Bag According to Published Recommendations

Transcription

1 Extractables Evaluation of a Single-Use Bioprocess Bag According to Published Recommendations April Chemic Laboratories Inc. 480 Neponset Street, Bldg. 7 Canton, MA

2 Study Objective An independent evaluation comparing various SUS extractable study parameters. To generate data sufficient to evaluate the merit of solvents, times, analytical techniques, and procedures. To evaluate and optimize analytical methodologies with a consideration toward extract compatibility. 2

3 Timeline (2015 Activities) 3

4 BPOG Study Design Solvent T=0 ( 30 min) at 25 C Time/Temperature 24 hours at 40 C 21 days at 40 C 30 days at 40 C 70 days at 40 C 50% EtOH Yes Yes Yes Yes Yes 1% PS-80 Yes Yes Yes Yes Yes 5M NaCl Yes Yes Yes Yes Yes 0.5N NaOH Yes Yes Yes Yes Yes 0.1M H 3 PO 4 Yes Yes Yes Yes Yes USP H 2 O Yes Yes Yes Yes Yes HEX Yes Yes Yes Yes Yes 100% EtOH Yes 100% IPA Yes 0.01M HCl Yes 38 extracts 370 unique assays 4 Instrumental Analysis Solvent HPLC-DAD/MS APCI +/- ESI +/- GC/MS HS-GC/MS ICP/MS 1 TOC ph NVR 1 50% EtOH Yes Yes Yes Yes Yes - Yes - 1% PS-80 Yes Yes Yes Yes Yes - Yes - 5M NaCl Yes Yes Yes Yes Yes Yes Yes - 0.5N NaOH Yes Yes Yes Yes Yes Yes Yes - 0.1M H 3 PO 4 Yes Yes Yes Yes Yes Yes Yes - USP H 2 O Yes Yes Yes Yes Yes Yes Yes Yes HEX Yes Yes Yes Yes 100% EtOH 1 Yes Yes Yes - Yes % IPA 1 Yes Yes Yes - Yes M HCl 1 Yes Yes Yes - Yes - Yes - 1 Only the final (70 day) time point was assessed.

5 Study Design Commercially available 1-liter 2D bioprocess bags were γ-irradiated at kgy and all associated components (ports, tubing, etc.) were removed and heat sealed with an internal wetted surface area of 726 cm2. Each test sample was filled with 121 ml of extraction solvent in order to achieve a 6:1 (cm2/ml) surface area: volume ratio. 5

6 Study Design Samples were prepared for each solvent & each time point, placed in secondary containment within an incubator. The study was staged in reverse within 5 weeks of γ-irradiation. At termination the sample extracts and controls were placed into inert containers for analytical testing. 6

7 Solvent Loss By ensuring the integrity of the sealing procedure and using secondary containment, solvent loss was not a significant concern. Solvent 24 hours at 40 C % Solvent Loss (w/w) 21 days at 40 C 30 days at 40 C 70 days at 40 C 50% EtOH 0.12% 2.6% 3.7% 8.7% 1% PS % 2.0% 2.6% 6.0% 5M NaCl 0.058% 1.1% 1.4% 3.3% 0.5N NaOH 0.078% 2.0% 2.4% 5.4% 0.1M H 3 PO % 2.0% 2.7% 5.2% USP H 2 O 0.087% 1.9% 2.6% 5.9% HEX 0.0% 1.8% 2.1% 5.2% 100% EtOH % 100% IPA % 0.01M HCl % 7

8 ph Results 8

9 NVR Results 70 day USP Water & Hexane extracts were transferred to appropriately cleaned vessels and evaporated to dryness. Description Extraction Solvent Mass Difference (initial vs. final) NVR (μg/cm 2 ) Method Control USP H 2 O NA Sample Extract USP H 2 O Method Control Hexane NA Sample Extract Hexane

10 TOC Results Notables Crystallization of glass Catalyst surface contaminant Devitrification of quartz and catalyst poisoning due to sodium content of extract solutions. Chemic has invested in 2 new state of the art TOC units, a combustion unit and a UV persulfate unit to resolve this issue. 10

11 TOC Results Quantitation was performed using external standardization. Linearity, precision, system suitability and internal QC met predetermined specifications. Description Extraction Solvent T=0 ( 30 min) at 25 C 24 hours at 40 C Results (μg/cm 2 ) 21 days at 40 C 30 days at 40 C 70 days at 40 C Sample Extract 5 M NaCl < LOQ < LOQ < LOQ < LOQ < LOQ Sample Extract 0.5 N NaOH < LOQ < LOQ < LOQ < LOQ < LOQ Sample Extract 0.1 M H 3 PO 4 < LOQ < LOQ < LOQ < LOQ < LOQ Sample Extract USP H 2 O The instrumental LOQ = 200 ppb ( 0.03 μg/cm2). Salt and base extract matrix effects resulted in a 1000X LOQ increase. 11

12 ICP/MS Results 70 day extracts were assayed for 30 metals via ICP/MS. Linearity, precision, system suitability and internal QC met predetermined specifications. Extraction Solvent Analyte Concentration (μg/cm 2 ) 50% EtOH ND 1% PS-80 ND 5M NaCl Copper N NaOH Copper M H 3 PO 4 ND USP H 2 O ND 100% EtOH ND 100% IPA ND 0.01M HCl ND 12

13 Qualitative Results All Organic solvents (mixtures) isolated a min. of 1 unique analyte whereas Aqueous solutions were not always successful. Solvent Number of extractable analytes across all time points (Number of analytes unique to extraction solvent) HPLC-DAD/MS DI-GC/MS HS-GC/MS 50% EtOH 9 (2) 4 (0) 14 (12) 1% PS (11) 2 (0) 2 (2) 5M NaCl 0 (0) 0 (0) 1 (1) 0.5N NaOH 10 (4) 1 (0) 0 (0) 0.1M H 3 PO 4 1 (0) 0 (0) 0 (0) USP H 2 O 0 (0) 0 (0) 0 (0) HEX 9 (6) 21 (8) % EtOH 12 (3) 12 (1) % IPA 8 (0) 16 (1) M HCl 1 (0) 0 (0)

14 HPLC-DAD/MS System Suitability Criteria Parameter Specification Result Standards BPA and Irganox Limit of Detection BPA, S:N 3:1 4.7:1-12.6:1 Precision 1 ppm BPA, RSD 20% (n=6) % BPOG Criteria 1ppm BPA in H2O; % % recovery Spike Recovery 1ppm BPA in 50% EtOH; % % recovery 2,4-di-tert-butylphenol Irganox Irganox Linearity R Irganox Irganox Chemic Criteria Irgafos BPA 1.00 Irganox Limit of Quantitation BPA, S:N 10:1, % recovery 21.7:1-48.3:1, % Accuracy 1 ppm BPA check std, % recovery % 14

15 HPLC-DAD/MS Results Notables The results indicated the presence of extractable peaks in all solvents with the exception of 5M NaCl and USP H2O. Linearity was run for eight common antioxidants establishing the instrumental range of the method from 0.1 to 2.5 μg/ml. 15

16 HPLC-DAD/MS Results Extraction Solvent 50% EtOH 1% PS-80 Analyte Concentration (μg/cm 2 ) (number of analytes) 30 Minutes 24 Hours 21 Days 30 Days 70 Days 2, 4-Di-tert-butylphenol (DAD; 280nm) ND bdtbpp (DAD; 280nm) ND Total unknowns ND (1) 0.32 (5) 0.17 (2) 0.65 (7) 2, 4-Di-tert-butylphenol (DAD; 280nm) ND bdtbpp (ESI-) ND ND Total unknowns 2.8 (1) 11.7 (4) 4.1 (3) 21 (6) 7.0 (6) 5 M NaCl None detected ND ND ND ND ND 0.5 N NaOH 2, 4-Di-tert-butylphenol (DAD; 280nm) ND ND ND Total unknowns ND 0.23 (1) 4.2 (2) 7.4 (6) 3.3 (7) 0.1 M H 3 PO 4 Total unknowns ND ND ND 0.04 (1) ND USP H 2 O None detected ND ND ND ND ND bdtbpp (ESI-) ND ND ND Hexane Total unknowns 4.8 (2) 7.1 (2) 11 (4) 3.0 (2) 3.6 (3) 2, 4-Di-tert-butylphenol (DAD; 280nm) % EtOH bdtbpp (DAD; 280nm) 0.11 Irgafos 168 phosphate (APCI+) 0.41 Total unknowns 3.5 (9) 2, 4-Di-tert-butylphenol (DAD; 280nm) % IPA bdtbpp (DAD; 280nm) 0.15 Irgafos 168 phosphate (APCI+) 0.47 Total unknowns 2.6 (5) 0.01 M HCl Total unknowns 0.16 (1) 16

17 DI-GC/MS System Suitability Criteria Parameter Specification Result Standards n-octane and BHT - Limit of BHT, S:N 3:1 2.0:1-3.6:1 Detection BPOG Criteria Precision 1 ppm BHT, RSD 20% (n=6) 4.7% (n=6) Chemic Criteria Spike Recovery 1 ppm BPA in H2O; % recovery % 1 ppm BPA in 1% PS-80; % recovery % Standards BHT - Linearity R BHT 1.00 Limit of Quantitation BHT, S:N 10:1, % recovery ~3.7:1-4.8:1, Accuracy 1 ppm BHT check (bracketing) standard, % recovery % 17

18 DI-GC/MS Results Notables The data demonstrated the presence of extractable peaks in all solvents with the exception of 5M NaCl, 0.1M H3PO4, USP H2O and 0.01M HCl. n-octane was initially evaluated and response was erratic due to its volatility. An extraction surrogate is added to monitor the efficiency of the liquid-liquid exchange procedure. The behavior of the surrogate may not be representative of all unknowns. 18

19 19 DI-GC/MS Results Extraction Solvent 50% EtOH 1% PS-80 Analyte Concentration (μg/cm 2 ) (number of analytes) 30 Minutes 24 Hours 21 Days 30 Days 70 Days 1,3-Di-tert-butylbenzene ,4-Di-tert-butylphenol ND Total siloxanes ND ND ND 0.03 (1) ND Total hydrocarbons ND ND ND 0.05 (1) ND 1,3-Di-tert-butylbenzene ,4-Di-tert-butylphenol ND M NaCl None detected ND ND ND ND ND 0.5 N NaOH 2,4-Di-tert-butylphenol ND M H 3 PO 4 None detected ND ND ND ND ND USP H 2 O None detected ND ND ND ND ND Hexane* 100% EtOH* 1,3-Di-tert-butylbenzene ,4-Di-tert-butylphenol Total siloxanes 0.22 (2) 0.06 (1) 0.63 (2) 0.24 (2) 0.56 (2) Total hydrocarbons 0.35 (8) 2.9 (16) 3.9 (17) 3.2 (17) 3.7 (17) 1,3-Di-tert-butylbenzene 3.3 2,4-Di-tert-butylphenol 0.48 Total siloxanes 0.22 (1) Total hydrocarbons 0.36 (9) 1,3-Di-tert-butylbenzene % IPA* 2,4-Di-tert-butylphenol 0.75 Total siloxanes 0.19 (1) Total hydrocarbons 0.96 (13) 0.01 M HCl None detected ND *Samples directly injected

20 Headspace-GC/MS System Suitability Criteria Parameter Specification Result Standards Methanol, MEK and D4 - Limit of Detection MEK, S:N 3:1 3.4:1 Precision 1 ppm MEK, RSD 20% (n=6) % (n=6) BPOG Criteria 1 ppm MEK in H 2 O; % Spike % recovery Recovery 1 ppm MEK in 1% PS-80; % recovery % Limit of Detection D4, S:N 3:1 4.4:1-5.6:1 1 ppm MEK check (bracketing) Accuracy Chemic Criteria standard, % recovery % Spike Recovery 1 ppm D4 in H 2 O; % recovery 96.6% 1 ppm D4 in 1% PS-80; % recovery 243% 20

21 HS-GC/MS System Suitability Notables Methanol was demonstrated to be unsuitable. Residual methanol in the ethanol further compounded the difficulty in interpretation. MEK and D4 spiked at 2.5 μg/ml were well resolved but displayed significant differences in relative response factors. Toluene-d8 is not water soluble and is may not be appropriate without added organic solvent modifier. System suitability criteria was met for MEK, however D4 recovery was not met in PS-80 due to co-elution. 21

22 HS-GC/MS Results Extraction Solvent 50% EtOH 1% PS-80 Analyte Concentration (μg/cm 2 ) (number of analytes) 30 Minutes 24 Hours 21 Days 30 Days 70 Days Methylcyclopentane ND ND ND ND 0.63 Cyclohexane ND ND ND ND 0.18 Diethoxyethane 0.04 ND ND Triethoxyborate ND ND ND ND 2.1 Octene ND ND ND ND 0.10 Octamethylcyclotetrasiloxane ND ND ND ND 0.23 Decamethylcyclopentasiloxane ND ND ND ND ,2-Di-tert-butylbenzene Total unknowns ND ND 0.02 (1) ND 0.59 (5) Octamethylcyclotetrasiloxane 0.18 ND ND ND ND 1,3-Di-tert-butylbenzene M NaCl tert-butanol ND ND 0.03 ND N NaOH None detected ND ND ND ND ND 0.1 M H 3 PO 4 None detected ND ND ND ND ND USP H 2 O None detected ND ND ND ND ND 22 Extraction Condition 3 grams dry film incubated at 40 C for 1 hour 3 grams dry film incubated at 80 C for 1 hour Number of Extractable Analytes Detected Representative Compound Classes Linear, cyclic and branched alkanes Linear and cyclic siloxanes Alcohols Ketones Silanols

23 General Study Conclusions Although limited in scope this study provides: -Additional information regarding aggressiveness of the solvents employed. -Insight into the variability that could be encountered. - Analytical challenges to be overcome associated with solvent-instrument compatibility. 23

24 General Study Conclusions cont. -Additional sample handling and analytical method optimization is required to ensure robust evaluation(s). -Significant investment is required to conduct studies to the level of rigor necessary to obtain usable data. 24

25 Key Facility Requirements Large chambers are required for a single study Typical TOC combustion units are not sufficient based on Na+ concentrations. All MS platforms must have dual ionization sources and ability to switch polarity +/-. A heat sealer is required to mitigate solvent loss from films. 25

26 Special Thanks To: Study plan reviewers (members of: BPOG, BPSA & USP and Industry SMEs) Test material supplier Chemic Laboratories analytical staff All others who have contributed to ongoing discussions For an expanded version of todays presentation visit: 26