extractables evaluation of a single-use bioprocess bag according to published recommendations

Chemic Laboratories Inc.480 Neponset Street, Bldg. 7Canton, MA 02021781-821-5600www.chemiclabs.com

Extractables Evaluation of a Single-Use Bioprocess Bag

According to Published Recommendations

April 2016

Copyright Chemic Laboratories Inc., 20161

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.


Timeline (2015 Activities)


Solvent

Time/Temperature

T=0 (≤ 30 min)

at 25°C

24 hours

at 40°C

21 days at 40°C

30 days at 40°C

70 days at 40°C

BP

OG

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 H3PO4 Yes Yes Yes Yes Yes

USP H2O Yes Yes Yes Yes Yes

HEX Yes Yes Yes Yes Yes

100% EtOH - - - - Yes

100% IPA - - - - Yes

0.01M HCl - - - - Yes

Study Design

SolventInstrumental Analysis

HPLC-DAD/MSGC/MS HS-GC/MS ICP/MS1 TOC pH NVR1

APCI +/- ESI +/-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 H3PO4 Yes Yes Yes Yes Yes Yes Yes -

USP H2O Yes Yes Yes Yes Yes Yes Yes YesHEX Yes Yes Yes - - - - Yes

100% EtOH1 Yes Yes Yes - Yes - - -100% IPA1 Yes Yes Yes - Yes - - -

0.01M HCl1 Yes Yes Yes - Yes - Yes -

38 extracts370 unique assays

1Only the final (70 day) time point was assessed.


Study Design

Commercially available 1-liter 2D bioprocess bags were γ-irradiated at 45-55 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.


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.


Solvent

% Solvent Loss (w/w)

24 hours at 40°C

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-80 0.093% 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 H3PO4 0.080% 2.0% 2.7% 5.2%

USP H2O 0.087% 1.9% 2.6% 5.9%

HEX 0.0% 1.8% 2.1% 5.2%

100% EtOH - - - 12%

100% IPA - - - 0.0%

0.01M HCl - - - 6.0%

Solvent Loss

By ensuring the integrity of the sealing procedure and using secondary containment, solvent loss was not a significant concern.


pH Results


NVR Results

DescriptionExtraction

SolventMass Difference (initial vs. final)

NVR (μg/cm2)

Method Control USP H2O -0.0003 NA

Sample Extract USP H2O 0.0003 0.93

Method Control Hexane 0.0001 NA

Sample Extract Hexane 0.0982 300

70 day USP Water & Hexane extracts were transferred to appropriately cleaned vessels and evaporated to dryness.


TOC Results Notables

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.


Crystallization of glass

Catalyst surface contaminant

TOC Results

DescriptionExtraction

Solvent

Results (μg/cm2)

T=0 (≤ 30 min)

at 25°C

24 hours at 40°C

21 days at 40°C

30 days at 40°C

70 days at 40°C

Sample Extract 5 M NaCl < LOQ < LOQ < LOQ < LOQ < LOQSample Extract 0.5 N NaOH < LOQ < LOQ < LOQ < LOQ < LOQSample Extract 0.1 M H3PO4 < LOQ < LOQ < LOQ < LOQ < LOQSample Extract USP H2O 0.034 0.13 0.23 0.61 0.72

Quantitation was performed using external standardization. Linearity, precision, system suitability and internal QC met predetermined specifications.

The instrumental LOQ = 200 ppb (≈ 0.03 μg/cm2). Salt and base extract matrix effects resulted in a 1000X LOQ increase.


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/cm2)

50% EtOH ND

1% PS-80 ND

5M NaCl Copper 2.2

0.5N NaOH Copper 0.06

0.1M H3PO4 ND

USP H2O ND

100% EtOH ND

100% IPA ND

0.01M HCl ND


Qualitative Results

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-80 14 (11) 2 (0) 2 (2)5M NaCl 0 (0) 0 (0) 1 (1)

0.5N NaOH 10 (4) 1 (0) 0 (0)

0.1M H3PO4 1 (0) 0 (0) 0 (0)

USP H2O 0 (0) 0 (0) 0 (0)

HEX 9 (6) 21 (8) - -100% EtOH 12 (3) 12 (1) - -100% IPA 8 (0) 16 (1) - -

0.01M HCl 1 (0) 0 (0) - -


All Organic solvents (mixtures) isolated a min. of 1 unique analyte whereas Aqueous solutions were not always successful.

13

HPLC-DAD/MS System Suitability

Criteria Parameter Specification Result

BPOG Criteria

Standards BPA and Irganox 1010 -

Limit of

DetectionBPA, S:N ≥ 3:1 4.7:1-12.6:1

Precision 1 ppm BPA, RSD ≤ 20% (n=6) 0.067-2.6%

Spike Recovery

1ppm BPA in H2O;

80-120% recovery101-102%

1ppm BPA in 50% EtOH;

80-120% recovery100-101%

Chemic Criteria

Linearity R2 ≥ 0.995

2,4-di-tert-butylphenol 0.993

Irganox 245 1.00

Irganox 259 1.00

Irganox 1010 1.00

Irganox 1076 1.00

Irgafos 168 1.00

BPA 1.00

Irganox 1010 1.00

Limit of

QuantitationBPA, S:N ≥ 10:1, 80-120% recovery 21.7:1-48.3:1, 96.2-101%

Accuracy1 ppm BPA check std, 80-120%

recovery93.8-102%


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.


HPLC-DAD/MS ResultsExtraction Solvent Analyte

Concentration (μg/cm2) (number of analytes)

30 Minutes 24 Hours 21 Days 30 Days 70 Days

50% EtOH

2, 4-Di-tert-butylphenol (DAD; 280nm)

ND 0.092 0.25 0.87 0.44

bDtBPP (DAD; 280nm) ND 0.032 0.05 0.08 0.07

Total unknowns ND 0.033 (1) 0.32 (5) 0.17 (2) 0.65 (7)

1% PS-80


ND 0.26 0.61 0.62 1.3

bDtBPP (ESI-) ND ND 1.1 1.3 0.14

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


ND ND ND 0.58 0.10

Total unknowns ND 0.23 (1) 4.2 (2) 7.4 (6) 3.3 (7)

0.1 M H3PO4 Total unknowns ND ND ND 0.04 (1) NDUSP H2O None detected ND ND ND ND ND

HexanebDtBPP (ESI-) ND ND ND 2.1 0.90

Total unknowns 4.8 (2) 7.1 (2) 11 (4) 3.0 (2) 3.6 (3)

100% EtOH


0.32

bDtBPP (DAD; 280nm) 0.11

Irgafos 168 phosphate (APCI+) 0.41

Total unknowns 3.5 (9)

100% IPA


0.46

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)


DI-GC/MS System Suitability


BPOG Criteria

Standards n-Octane and BHT -

Limit of

DetectionBHT, S:N ≥ 3:1 2.0:1-3.6:1

Precision 1 ppm BHT, RSD ≤ 20% (n=6) 4.7% (n=6)

Spike Recovery1 ppm BPA in H2O; 80-120% recovery 96.8-107%

1 ppm BPA in 1% PS-80; 80-120% recovery 96.6-106%

Chemic Criteria

Standards BHT -

Linearity R2 ≥ 0.995 BHT 1.00

Limit of

QuantitationBHT, S:N ≥ 10:1, 80-120% recovery ~3.7:1-4.8:1, 90.6-94.1

Accuracy1 ppm BHT check (bracketing) standard,

80-120% recovery84.5-116%


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”.


DI-GC/MS ResultsExtraction Solvent Analyte

Concentration (μg/cm2) (number of analytes)


50% EtOH

1,3-Di-tert-butylbenzene 0.06 0.19 0.15 0.14 0.34

2,4-Di-tert-butylphenol ND 0.027 0.10 0.50 0.35

Total siloxanes ND ND ND 0.03 (1) NDTotal hydrocarbons ND ND ND 0.05 (1) ND

1% PS-801,3-Di-tert-butylbenzene 0.02 0.06 0.08 0.11 0.11

2,4-Di-tert-butylphenol ND 0.04 0.05 0.20 0.44

5 M NaCl None detected ND ND ND ND ND

0.5 N NaOH 2,4-Di-tert-butylphenol ND 0.04 0.05 0.05 0.02

0.1 M H3PO4 None detected ND ND ND ND NDUSP H2O None detected ND ND ND ND ND

Hexane*

1,3-Di-tert-butylbenzene 1.6 2.6 2.3 3.0 3.52,4-Di-tert-butylphenol 0.06 0.05 0.64 0.21 0.36

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)

100% EtOH*

1,3-Di-tert-butylbenzene 3.3

2,4-Di-tert-butylphenol 0.48

Total siloxanes 0.22 (1)

Total hydrocarbons 0.36 (9)

100% IPA*

1,3-Di-tert-butylbenzene 2.8

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 injectedCopyright Chemic Laboratories Inc., 201619

Headspace-GC/MS System Suitability


BPOG Criteria

Standards Methanol, MEK and D4 -

Limit of

DetectionMEK, S:N ≥ 3:1 3.4:1

Precision 1 ppm MEK, RSD ≤ 20% (n=6) 7.9-11% (n=6)

Spike

Recovery

1 ppm MEK in H2O;

70-130% recovery76.4-108%

1 ppm MEK in 1% PS-80;

70-130% recovery96.2-114%

Chemic Criteria

Limit of

DetectionD4, S:N ≥ 3:1 4.4:1-5.6:1

Accuracy1 ppm MEK check (bracketing)

standard, 70-130% recovery75.0-118%

Spike

Recovery

1 ppm D4 in H2O; 70-130% recovery 96.6%

1 ppm D4 in 1% PS-80;

70-130% recovery243%


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.


HS-GC/MS Results

Extraction Solvent AnalyteConcentration (μg/cm2) (number of analytes)


50% EtOH

Methylcyclopentane ND ND ND ND 0.63

Cyclohexane ND ND ND ND 0.18

Diethoxyethane 0.04 ND ND 0.02 0.34

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 0.04


Total unknowns ND ND 0.02 (1) ND 0.59 (5)

1% PS-80Octamethylcyclotetrasiloxane 0.18 ND ND ND ND


5 M NaCl tert-Butanol ND ND 0.03 ND 0.03

0.5 N NaOH None detected ND ND ND ND ND0.1 M H3PO4 None detected ND ND ND ND ND

USP H2O None detected ND ND ND ND ND

Extraction ConditionNumber of Extractable Analytes

DetectedRepresentative Compound Classes

3 grams dry film incubated at 40°C

for 1 hour29

Linear, cyclic and branched alkanesLinear and cyclic siloxanes

AlcoholsKetonesSilanols

3 grams dry film incubated at 80°C

for 1 hour45


General Study Conclusions

Although limited in scope this study provides:

-Additional information regarding aggressiveness of thesolvents employed.

-Insight into the variability that could be encountered.

- Analytical challenges to be overcome associatedwith solvent-instrument compatibility.


General Study Conclusions cont.

-Additional sample handling and analytical methodoptimization is required to ensure robust evaluation(s).

-Significant investment is required to conduct studiesto the level of rigor necessary to obtain usable data.


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.


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 presentationvisit: http://www.chemiclabs.com/publications.php

26 Copyright Chemic Laboratories Inc., 2016

http://www.chemiclabs.com/publications.php

extractables evaluation of a single-use bioprocess bag according to published recommendations

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