Microbial Survival in Compressed Gases Under Fast Decompression to Normal Atmospheric Conditions Background Microbiological gas testing systems are commercially available. Monitoring compressed process gases and air in direct contact with pharmaceutical drugs during manufacturing: • is vital to the quality and safety of the product, and • a key requirement under GMP regulations for aseptic manufacturing. The sampling methodology used for compressed gases involves the sudden decompression of compressed gas right before taking a sample for microbial counting. Issue • It has been proposed that asserts that the decompression step could significantly deteriorate microbial cells and affect their ability to generate colony forming units 1 . This speculation is proven false through a review of regulations, applications and the numerous scientific literature related to microbial survival under a broad range of environmental pressure conditions. Facts In pharmaceutical environments, microorganisms are exposed to pressure reductions: • Typically from 2.5 to 1.1 bar • A maximum of 10 bar decreased to 1.1 bar in a worst-case scenario Microorganisms are resilient under variable compression and decompression environments. Gas Applications Used in Pharmaceuticals and Biotechnology Cryopreservation & Lyophilization Liquid Nitrogen, Cryogenic Storage Solutions Aseptic Packaging Nitrogen Pressure & Leak Testing Carbon Dioxide, Helium, Nitrogen Process Chilling Liquid Carbon Dioxide, Liquid Nitrogen Purging Carbon Dioxide, Nitrogen Wastewater Treatment Oxygen Blanketing Carbon Dioxide, Nitrogen ph Control Carbon Dioxide Pressure Transfer Nitrogen Filtration, Separation & Purification Variety of CG Analysis & Instrumentation CG Mixtures a review by: Frank Panofen, Particle Measuring Product Line Manager - Sterility Assurance , Microbiology Microbes Survive Under Compression and Decompression Microbes are not humans Instrumentation Selection Instrumentation Financials and Return of Investment (ROI) Microbial cells survive the volatile external pressures endured in multiple harsh environments, including extreme deep-sea environments. 7 Microbes conserve the properties necessary for life to originate under harsh conditions. 6 Bacterial cultures in food products require 2500- 3000 bar * to inactivate 3 Cells treated at pressures of <1000 bar * showed no significant loss of viability 3 Lessened microbial growth & viability on metabolic processes in vivo are seen with pressures higher than 1000 bar* 5 Serratia and Carnobacterium spp survive conditions similar to Mars (low O2, <0°C, 7 mbar) 5 Findings from high-pressure studies: • 92% recovery using the MAS100 CG • Bacterial cells maintained suitable growth, even up to 10 bar during the incubation. • Compression cycle did not influence the viability of the tested microorganisms. • Hypothesis from study: sampling prior to decompression avoided potential microbial cell damage and lowered viability. This hypothesis is refuted by multiple studies. • Compare to decompression from 10 to 1 bar found in a CG sampler Decompression-sensitive bacteria study • Size and weight - Liſting heavy instruments to a sampling point or pushing a huge enclosure below a filling line should be minimized. • Preferred usage of mobile instruments/handhelds • Ease of use • Soſtware/data management - GMP compliance • Cleaning/disinfection - No contribution to the particle load Rare measurement vs. investment Frequency of monitoring compressed gases • Classification: Performed monthly (1-6 months) or quarterly (6-12 months) • Routine testing: EU-2x year, US-1x year Testing is not frequent and investing into dedicated equipment with all associated costs like validation, maintenance and repair, is difficult to financially justify. Alternatives to dedicated instrument investment Option 1 - Service Provider Sample scheduling and control of data and measurements can become difficult without quality communication and agreements between parties. Option 2 - Multipurpose Instrument Instruments are readily available and their validation maintained due to scheduled maintenance and, if necessary, repair. Additional costs only from the accessory. A comparison of bacterial recovery from compressed gases using the MAS100 CG and a standard air filter method found 1 : Escherichia coli and Corynebacterium xerosis survive rapid decompression from 300 bar Cells without gas vesicles were not harmed by decompression from up to 300 bar .4 0 50 bar 100 bar 150 bar 200 bar 250 bar 300 bar 300 bar | Escherichia coli and Corynebacterium xerosis survive 10 bar | 10 to 1 bar found in a CG sample Gram-negative, gas vacuolate bacteria (M. aquaticus, P. pneumaticum, and M. glaucopis) are known to be very susceptible to decompression. 4 • Populations of these cells were saturated with Ar, N 2 or He up to 100 bar. Gas phases of the vesicles remained intact. • Upon rapid decompression to atmospheric pressure, the vesicles expanded and ruptured the cells. • Viable counts indicated minimum pressures were between 25 - 50 bar. • Majority of the cell envelopes were ruptured at pressures between 50 - 100 bar. Decompression of gases has no influence on the viability of microorganisms. Instrument selection is based on the following parameters: 1 Sandle, T. (2015). Microbiological Assessment of Compressed Gases in Pharmaceutical Facilities. Journal of Validation Technology. Institute of Validation Technology (IVT Network.com). 2 Zingre, H. and Meier, R. (2013). Detection of Microorganisms in Compressed Gases. MBV AG Application Note. Stäfa, Switzerland. 3 FDA. (2014). Kinetics of Microbial Inactivation for Alternative Food Processing Technologies - High Pressure Processing (HPP). A report of the Institute of Food Technologists for the Food and Drug Administration of the U.S. Department of Health and Human Services. Updated December 2014. 4 Hemmingsen, B. B. & Hemmingsen, E. A. (1980). Rupture of the Cell Envelope by Induced Intracellular Gas Phase Expansion in Gas Vacuolate Bacteria. Journal of Bacteriology. 143 no. 2:841-846. 5 Nicholson, W. (2013). Growth of Carnobacterium spp. from permafrost under low pressure, temperature, and anoxic atmosphere has implications for Earth microbes on Mars. pnas 110(2):666- 671. 6 Picard A., & Daniel I. (2013). Pressure as an environmental parameter for microbial life–a review. Biophysical Chemistry. 183, 30-41 PMID: 23891571. 7 ALVIN. (2016). Project ALVIN Life around deep sea vents. http://www.amnh.org/explore/curriculum-collections/deep-sea-vents/pressure-in-the-deep-seas/ American Museum of Natural History. Summary Sampling aſter decompression parallels a typical scenario of using compressed gases during different stages of the manufacturing process. Microorganisms under hyperbaric or hydrostatic pressures ranging from 1 to 10 bar resulted in no significant reduction of viability. Decompression from 10 bar to normal atmospheric pressure does not harm microorganisms. Decompression occurring before or aſter agar plate impaction does not decrease viability and recovery of CFUs. “...sampling device shall be selected according to the area being monitored. The selection for a particular application shall take into consideration the following factors… effect of the sampling device on the process or environment to be monitored …” ISO 14698 7 MiniCapt® Mobile, PMS Particle Measuring Systems MAS-100 CG, MBV AG RCS High Flow, MerckSigm *1 Bar = 0.1 Mpa = 14.5 PSI
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Microbial Survival in Compressed Gases Under Fast Decompression to Normal Atmospheric Conditions
BackgroundMicrobiological gas testing systems are commercially available.
Monitoring compressed process gases and air in direct contact with pharmaceutical drugs during manufacturing:
• is vital to the quality and safety of the product, and
• a key requirement under GMP regulations for aseptic manufacturing.
The sampling methodology used for compressed gases involves the sudden decompression of compressed gas right before taking a sample for microbial counting.
Issue• It has been proposed that asserts that the
decompression step could significantly deteriorate microbial cells and affect their ability to generate colony forming units1.
This speculation is proven false through a review of regulations, applications and the numerous scientific literature related to microbial survival under a broad range of environmental pressure conditions.
FactsIn pharmaceutical environments, microorganisms are exposed to pressure reductions:
• Typically from 2.5 to 1.1 bar
• A maximum of 10 bar decreased to 1.1 bar in a worst-case scenario
Microorganisms are resilient under variable compression and decompression environments.
Gas Applications Used in Pharmaceuticals and BiotechnologyCryopreservation & Lyophilization Liquid Nitrogen, Cryogenic Storage Solutions
Aseptic PackagingNitrogen
Pressure & Leak TestingCarbon Dioxide, Helium, Nitrogen
Process ChillingLiquid Carbon Dioxide, Liquid Nitrogen
PurgingCarbon Dioxide, Nitrogen
Wastewater TreatmentOxygen
BlanketingCarbon Dioxide, Nitrogen
ph ControlCarbon Dioxide
Pressure TransferNitrogen
Filtration, Separation & PurificationVariety of CG
Analysis & InstrumentationCG Mixtures
a review by: Frank Panofen, Particle Measuring Product Line Manager - Sterility Assurance , Microbiology
Microbes Survive Under Compression and Decompression
Microbes are not humans
Instrumentation Selection Instrumentation Financials and Return of Investment (ROI)
Microbial cells survive the volatile external pressures endured in multiple harsh environments, including extreme deep-sea environments.7
Microbes conserve the properties necessary for life to originate under harsh conditions.6
Bacterial cultures in food products
require 2500- 3000 bar* to inactivate3
Cells treated at pressures of <1000
bar* showed no significant loss of
viability3
Lessened microbial growth & viability on metabolic processes
in vivo are seen with pressures higher
than 1000 bar*5
Serratia and Carnobacterium spp survive conditions
similar to Mars (low O2, <0°C, 7 mbar)5
Findings from high-pressure studies:
• 92% recovery using the MAS100 CG• Bacterial cells maintained suitable growth, even up to 10 bar during
the incubation.• Compression cycle did not influence the viability of the tested
microorganisms.• Hypothesis from study: sampling prior to decompression avoided
potential microbial cell damage and lowered viability. This hypothesis is refuted by multiple studies.
• Compare to decompression from 10 to 1 bar found in a CG sampler
Decompression-sensitive bacteria study
• Size and weight - Lifting heavy instruments to a sampling point or pushing a huge enclosure below a filling line should be minimized.
• Preferred usage of mobile instruments/handhelds
• Ease of use
• Software/data management - GMP compliance
• Cleaning/disinfection - No contribution to the particle load
Rare measurement vs. investmentFrequency of monitoring compressed gases• Classification: Performed monthly (1-6 months) or quarterly
(6-12 months)• Routine testing: EU-2x year, US-1x yearTesting is not frequent and investing into dedicated equipment with all associated costs like validation, maintenance and repair, is difficult to financially justify.
Alternatives to dedicated instrument investmentOption 1 - Service Provider
Sample scheduling and control of data and measurements can become difficult without quality communication and agreements between parties.
Option 2 - Multipurpose InstrumentInstruments are readily available and their validation maintained due to scheduled maintenance and, if necessary, repair. Additional costs only from the accessory.
A comparison of bacterial recovery from compressed gases using the MAS100 CG and a standard air filter method found1:
Escherichia coli and Corynebacterium xerosis survive rapid decompression from 300 bar
Cells without gas vesicles were not harmed by decompression from up to 300 bar.4
0
50 bar
100 bar
150 bar
200 bar
250 bar
300 bar 300 bar | Escherichia coli and Corynebacterium xerosis survive
10 bar | 10 to 1 bar found in a CG sample
Gram-negative, gas vacuolate bacteria (M. aquaticus, P. pneumaticum, and M. glaucopis) are known to be very susceptible to decompression.4
• Populations of these cells were saturated with Ar, N2 or He up to 100 bar. Gas phases of the vesicles remained intact.
• Upon rapid decompression to atmospheric pressure, the vesicles expanded and ruptured the cells.
• Viable counts indicated minimum pressures were between 25 - 50 bar.• Majority of the cell envelopes were ruptured at pressures between
50 - 100 bar.
Decompression of gases has no influence on the viability of microorganisms. Instrument selection is based on the following parameters:
1 Sandle, T. (2015). Microbiological Assessment of Compressed Gases in Pharmaceutical Facilities. Journal of Validation Technology. Institute of Validation Technology (IVT Network.com).
2 Zingre, H. and Meier, R. (2013). Detection of Microorganisms in Compressed Gases. MBV AG Application Note. Stäfa, Switzerland.
3 FDA. (2014). Kinetics of Microbial Inactivation for Alternative Food Processing Technologies - High Pressure Processing (HPP). A report of the Institute of Food Technologists for the Food and Drug Administration of the U.S. Department of Health and Human Services. Updated December 2014.
4 Hemmingsen, B. B. & Hemmingsen, E. A. (1980). Rupture of the Cell Envelope by Induced Intracellular Gas Phase Expansion in Gas Vacuolate Bacteria. Journal of Bacteriology. 143 no. 2:841-846.
5 Nicholson, W. (2013). Growth of Carnobacterium spp. from permafrost under low pressure, temperature, and anoxic atmosphere has implications for Earth microbes on Mars. pnas 110(2):666- 671.
6 Picard A., & Daniel I. (2013). Pressure as an environmental parameter for microbial life–a review. Biophysical Chemistry. 183, 30-41 PMID: 23891571.
7 ALVIN. (2016). Project ALVIN Life around deep sea vents. http://www.amnh.org/explore/curriculum-collections/deep-sea-vents/pressure-in-the-deep-seas/ American Museum of Natural History.
SummarySampling after decompression parallels a typical scenario of using compressed gases during different stages of the manufacturing process.
Microorganisms under hyperbaric or hydrostatic pressures ranging from 1 to 10 bar resulted in no significant reduction of viability.
Decompression from 10 bar to normal atmospheric pressure does not harm microorganisms.
Decompression occurring before or after agar plate impaction does not decrease viability and recovery of CFUs.
“...sampling device shall be selected according to the area being monitored. The selection for a particular application shall take into consideration the following factors… effect of the sampling device on the process or environment to be monitored …” ISO 146987
MiniCapt® Mobile, PMS Particle Measuring Systems MAS-100 CG, MBV AG
RCS High Flow, MerckSigm
*1 Bar = 0.1 Mpa = 14.5 PSI
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